Abstract

Since the industrial Revolution, as the global economy has boomed and the agricultural population has expanded. Excessive use of chemical fertilizer and unreasonable farming methods make soil salinization more and more serious. Chenopodium quinoa, it has unique nutritional value and strong stress resistance and adaptability, under the background of soil salinization, quinoa has been widely studied as a halophyte model. With the release of high-quality genome of quinoa, more and more salt-tolerant genes of quinoa have been cloned gradually. Bioinformatics and expression analysis of GST gene in quinoa in this study, 114 CqGST genes were identified from the whole genome of quinoa by bioinformatics methods. The phylogenetic tree showed that 114 CqGST genes were divided into seven subgroups: GSTU (68 members), GSTF (23 members), GST members), GSTZ (6 members), GSTT (5 members), DHAR (4 members) and TCHQD (2 members). Gene structure and Motif analysis showed high similarity among members of each subgroup. Phylogenetic analysis of these genes suggested that tandem and fragment replication events played a key role in the expansion of the CqGSTs gene family, and the CqGST genes may have undergone strong purification selection during the evolution process. Analysis of salt-treated transcriptome from the roots of salt-tolerant and salt-sensitive quinoa cultivars showed that Salt treatment induced changes in the expression levels of CqGSTs genes, and eight CqGST genes (CqDHAR2, CqDHAR3, CqGSTU22, CqGSTU44, CqGSTU60, CqGSTU63, CqGSTU67, CqGSTU68) were steadily up-regulated in both cultivars. RT-qPCR results showed that these selected CqGST genes were not only induced by salt stress, but also by drought stress.

1. Introduction

Quinoa (Chenopodium quinoa Willd), a heterologous tetraploid (2n=4x=36), is an annual crop of Amaranthus family, with a genome size of about 1.5 Gbp ¹. Quinoa grains are nutritionally rich and balanced, rich in protein, starch, VB1, folic acid, minerals (Ca, Zn, Fe) and other nutrients ². In addition, because quinoa grows at high altitude (> 3500 meters above sea level) all year round, it is subjected to drought, low temperature, salt and other abiotic stresses during its growth and development, which not only limits the planting area, but also affects the growth, development and yield of quinoa ³. With the publication of high-quality reference genomes ⁴. Quinoa has now become an important plant material for studying the mechanism of salt tolerance in plants. Basic helix-loop-helix (bHLH) family is the second largest gene family in plants, and is involved in many biological processes such as plant growth, development, metabolism, and resistance to abiotic stress ⁵. However, this gene family has not been identified in Quinoa. The systematic identification of quinoa bHLH gene family members in quinoa genome, and the analysis of their chromosome location, gene structure, evolutionary relationship and expression characteristics are of great significance to elucidate the response of quinoa bHLH transcription factor family to salt stress, which is of great significance to enrich the mechanism of salt tolerance of quinoa and breed new varieties of quinoa tolerant to salt stress. The bHLH transcription factor family contains two highly conserved domains. The alkaline domain is located at the amino terminus of bHLH and is responsible for binding to specific DNA sequences and recognizing E-box elements ⁶. The bHLH region is located at the carboxyl terminal and is connected by a ring structure of two alpha-helices and hydrophobic amino acids, containing 40 amino acids ⁷. Due to the flexibility of helices, the bHLH domain facilitates protein interactions and regulates the expression of target genes through the formation of homodimer or heterodimer complexes ⁸. According to the conserved and structural characteristics of the gene sequences, the Arabidopsis family is divided into 21 subfamilies ⁹. It has been reported that the bHLH transcription factor family is mainly involved in abiotic stress in plants, such as in response to drought, low temperature, salt, and abscisic acid hormones ^{10, 11}. In Arabidopsis thaliana, AtbHLH006, AtbHLH17, AtbHLH32, AtbHLH92, AtbHLH122, AtbHLH128 and AtbHLH130 directly or indirectly participate in abscisic acid signaling pathway to improve drought resistance of Arabidopsis thaliana ¹². Overexpression of bHLH transcription factors MYC type ICE1, ICE2 and CBF enhanced Arabidopsis tolerance to low temperature stress ¹³. In wheat, TabHLH1 can regulate abscisic acid mediated stress tolerance pathway, thereby improving plant adaptability to drought and salt stress ¹⁴. TabHLH39 gene is involved in regulating gene expression in response to stress, thereby improving salt tolerance of overexpressed wheat plants ¹⁵. In rice, OsbHLH148 and OsbHLH006 (RERJ1) respond to drought stress through jasmonic acid signaling pathway ^{16, 17}. In the process of drought and abscisic acid induction, the expression of PebHLH35 gene was increased in populus euphratica, which actively participated in the regulation of drought stress, thus improving the tolerance of populus euphratica ¹⁸. And has been found to have different degrees of functional differentiation. Although the whole genome of Quinoa was sequenced by Jarvis DE et al in 2017 ¹⁹. An annual dicotyledonous plant that originated in the Andes and has been cultivated for about 5,000 to 7,000 years, it was the main food crop of the local Inca people ²⁰. Quinoa seeds are rich in nutrients and are considered an international food of the United Nations Agriculture Organization (FAO) praised as the only "whole nutrition food" that can meet the basic nutrition of human body with all kinds of single plants ²¹. Therefore, the general attention of the people, the output shows multiple growth. As a halophytes ²². Quinoa is drought - tolerant, salt - alkali - tolerant and barren - resistant. With the publication of a high-quality reference genome for Quinoa ²³. The Plants have become one of the important materials for studying the mechanism of plant stress resistance. Quinoa is mainly distributed in Peru, Bolivia, Ecuador and Chile, and is native to South America the Andes Mountains region of Asia is the traditional food crops of the Inca indigenous people, which has been about 5000~7000 years ago Years of planting history ^{24, 25}. Quinoa is grown widely in South America, from 2° north latitude in Colombia to southern Chile. It can be cultivated from sea level to 4,000 m above sea level at latitude 40° ^{26, 27}. Because quinoa is also exposed to extreme environmental conditions it still has a high yield ²⁸. The discovery of special nutritional properties of quinoa, health care based on quinoa as a raw material Products and food are more and more consumers love, relying solely on the country of origin production in short supply. Quinoa leaves are broad with serrated edges, smooth or palmately divided margins, and generally have short velvet Hairy, annual dicotyledonous plants, single leaves alternate, depending on the variety of plant height, roots, stems, leaves, flowers, fruit And other plant characteristics, with the characteristics of genetic diversity. Quinoa has a strong resistance and adaptability, can be in the dry It grows in extremely harsh environment such as drought, salinity and frost, and is suitable for growing in arid, salinized and other marginal areas ^{29, 30}. Quinoa was native to high altitudes of several thousand meters. In order to adapt to the harsh environment, quinoa has a net root system like distribution, very developed, this structure helps to resist drought, strong wind and barren environment ^{31, 32}. The smaller leaf area and the abundant vesicle structure on the leaf surface of Quinoa are beneficial to reduce transpiration and thus resist drying arid environment ^{33, 34}. Physiological, quinoa mainly through the accumulation of proline, betaine, soluble sugar and other organic as well Inorganic permeates form tissue elasticity to achieve osmotic regulation. Quinoa also relies on its vesicle structure to accumulate salt tolerance Organic osmotic regulators such as proline, betaine, polyamine and dehydrating protein can remove reactive oxygen species in vivo ³⁵. Overaccumulation of osmotic regulating substances, Na+ efflux and K+ retention achieve osmotic balance of inorganic ions. Dehydrin and the accumulation of soluble sugars explained the frost resistance of quinoa from the physiological level ³⁶. In this study, bioinformatic methods were used to identify the quinoa bHLH transcription factor family, and their bioinformatic characteristics such as distribution, gene structure, evolutionary differentiation, tissue expression, and salt-stress-induced expression specificity were systematically analyzed, laying a foundation for clarifying the differentiation process and biological function of quinoa bHLH transcription factor family. Phylogenetic analysis of these genes suggested that tandem and fragment replication events played a key role in the expansion of the CqGSTs gene family, and the CqGST genes may have undergone strong purification selection during the evolution process.

2. Materials and Methods

The experiment was conducted in the laboratory of Wheat Research Station, College of Agronomy, Shanxi Agricultural University during 2021-2022.

Quinoa variety Faro was the experimental material in this chapter. Quinoa seeds were placed in sterilized camps culture in soil. The indoor culture temperature was 23°C and photoperiod was 16 h light / 8 h darkness. In order to study CqGST gene expression patterns under different stress, one-month old Quinoa seedlings with the same growth rate were selected, and 60 mL were used, respectively. The treatment was treated with 300 mmol/L NaCl and 20% PEG6000 solution for 3, 6, 12, 24 and 48 h. Acquisition and processing the above-ground and underground parts of the seedlings were immediately frozen in liquid nitrogen and stored in an ultra-low temperature refrigerator at −80℃ for subsequent analysis, each treatment was repeated three times.

According to the quinoa genome database GST gene sequence information, use the Primer - BLAST (https:// www.ncbi.nlm.nih.gov/tools/primer-blast/) online website design qRT - PCR primers used to detect the base due to the expression in different treatments and different treatment times, the primer sequence is shown in Table 1.

The 50×TAE electrophoresis buffer (1L): Weigh 242g of Tris in an electronic balance and stir with appropriate amount of deionized water Mix and dissolve, add 57.1 mL glacial acetic acid, add 0.5 mol EDTA (pH 8.0) 100 mL, constant volume to 1L.

From arabidopsis thaliana TAIR database download known arabidopsis Jsp (https://www.arabidopsis.org/index.). Mustard GST protein sequence, known rice downloaded from the National Rice Data Center (https://www.ricedata.cn/). The sequence of the quinoa genome was downloaded from Chenopodium DB (h ttps://www.cbrc.kaust.edu.sa/chenopodiumdb/). Based on known GST sequences in Arabidopsis thaliana and rice. The TBTools tool kit was used to compare the data in the quinoa genome database, and the e value was set as E-10. The use CDD NCBI database (https://www.ncbi.nlm.nih.gov/cdd/) and SMART (https://smart.embl-heidelberg.de) database was used to screen quinoa GST genes with conserved domains gST-C and gST-N NCBI database (https://www.ncbi.nlm.nih.gov/) to verify the accuracy of the results. According to quinoa G the relationship between ST gene and Arabidopsis GST gene and the position of quinoa GST gene on chromosome were investigated Name it.

The genome sequence and gene structure annotation of Quinoa used in the study were downloaded from the NCBI Assembly database (Accession number: PI614886, Biosample accession: SAMN04338310) ³⁷. Using the published protein sequences of the bHLH family members of Arabidopsis ³⁸ and rice ¹⁹ as reference sequences, local BLastPs were constructed by Tbtools, and homologous alignment was performed in the protein sequence library of Quinoa, with E value < 1E-10. In addition, the hidden Markov (HMM) model of characteristic conserved domain bHLH (PF00010) was downloaded from the Pfam database, and the "hmm search" program of Hmmer 3.0 software was used to conduct homology search in the quinoa protein sequence library, and E value < 1E-5 was set. The results of the above two methods are combined to obtain non-redundant candidate protein sequences. Using NCBI-CDD database (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi) and SMART database (https://smart.embl-heidelberg.de/) on the above candidate eggs the white sequences were searched for conserved domains to determine that the candidate proteins contained the bHLH domain. Finally, by removing incomplete sequences and redundant protein sequences, high confidence members of the quinoa bHLH gene family were identified.

Based on the gene structure annotation file, the sequence length, chromosome localization, encoding protein length and intron number of the bHLH gene of Quinoa were sorted out. Using Expasy online tools (https://web.expasy.org/compute_pi/) to calculate the quinoa bHLH proteins isoelectric point (pI) and molecular weight (Mw). Use of Cell PLoc 2.0 online tools (https://www.csbio.sjtu.edu.cn/bioinf/plant-multi/) to predict the subcellular localization of the members of the family. The hydrophilicity of quinoa bHLH protein coefficient calculated by ProtScale online tools (https://web.expasy.org/protscale/).

Expression data of CqbHLH gene in the root, stem and leaf of Quinoa (Accession number: SRP278144) were obtained from NCBI-SRA database and used for expression pattern analysis. Expression data of CqbHLH gene in salt-tolerant and salt-sensitive cultivars of quinoa (Accession number: SRP247883) were obtained from NCBI-SRA database and used for salt-tolerant expression pattern analysis. Tbtools was used to draw the heat map of expression patterns, and the expression level Log2 (FPKM) of the gene family members in different tissues was standardized. Euclidean-style distance method was used to cluster genes with similar expression patterns. The database and website used in this experiment are as follows:Chenopodium DB (https://w) Ww.cbrc.kaust.edu.sa/chenopodiumdb/) Arabidopsis thaliana database (https://www.arabidopsis.org/); National Rice Data Center (https://www.ricedata.cn/); NCBI (https://www.ncbi.nlm.nih.gov/). NCBI CDD (https://www.ncbi.nlm.nih.gov/cdd/); SMART (https://smart.embl-heidelb) erg.de).

The online website and software involved in the bioinformatics analysis of this experiment are as follows: Evolview (https://evolge nius.info//evolview-v2/); MEME (https://memesuite.org/tools/meme).ExPASy(https://web.expasy.org/); CELLO (https://cello.life.nctu.edu.tw/); Plant CARE(https://biOinformatics.PSB.Ugent.Be/webtools/plantcare/HTML /); MEGA6.0; TB Tools; Excel 2016.

Use ExPASy online website (https://www.Expasy.ch/tools/pi_tool.html) for analysis and identification Amino acid number, molecular weight, isoelectric point, instability index, fat index and hydrophilicity of quinoa GST protein Isophysical and chemical properties. Use the CELLO (https://cello.life.nctu.edu.tw/) online prediction quinoa GST gene subcellular localization.

The MEGA6.0 software was used to compare the full-length sequences of GSTs in quinoa, Arabidopsis and rice the phylogenetic evolutionary tree was constructed by likelihood method, and the bootstrap value was set to 1000. With Evolview (https://evol genius.info//evolview-v2/). Visual analysis of phylogenetic trees on online website.

The gene structure information of Quinoa GSTs was extracted from the gene annotation information of Quinoa reference genome. Will identify. The protein sequence of quinoa GSTs was submitted to MEME (https://meme-suite.org/tools/meme). Row motif search and identification, the upper limit of the conserved domain is set to 10, and the conserved domain is allowed to repeat. The phylogeny, gene structure and conserved motifs of Quinoa were analyzed using TBtools.

The distribution of Quinoa GST gene on chromosomes was mapped using TBtools. Using the McScanx method. The replication modes of quinoa GST gene were divided into tandem replication and segmenial replication. Ka/Ks are calculated using TBtools toolkit. To understand collinearity between homologous GST genes in Quinoa and other species, the TBtools toolkit was used to analyze the data Visualization is performed.

Microsoft Excel 2016 was used to sort out the original data, Origin 8.5 and Graphpad were adopted. Analysis of variance and significance test were performed by prism 8 software.

3. Results

A total of 250 CqbHLH transcription factor family members were identified from the whole genome of Quinoa using bioinformatics methods and subsequent screening verification, which were successively named CqbHLH1-CqbHLH250 according to chromosome sequence. 237 CqbHLH transcription factors were randomly distributed on all 18 chromosomes. There are 6 to 27 transcription factors per chromosome, of which 13 transcription factors (CqbHLH238-CqbHLH250) are not anchored to the chromosome (Table 2). The amino acid length of the CqbHLH transcription factor family was between 83(CqbHLH243) and 962(CqbHLH100). The molecular weight ranged from the minimum 8.90kD (CqbHLH243) to the maximum104.51kD (CqbHLH100). Isoelectric points ranged from 4.49 (CqbHLH112) to 11.84 (CqbHLH107), of which 173 were below 7 (Table 2). Therefore, the length span of quinoa bHLH transcription factor family genes was large and correlated with molecular weight, and 157 transcription factors were located in the nucleus. The total average of hydrophilicity (GRAVY) of all CqbHLH transcription factors is positive, indicating that all CqbHLH transcription factors are likely to be soluble proteins, which is in line with functional positioning as transcription factors. By comparing the sequences of the bHLH gene family members of Arabidopsis thaliana and the bHLH transcription factor family members of Quinoa, it can be seen that most of the bHLH transcription factors of quinoa have high homology with that of Arabidopsis thaliana (Table 2), which provides a reference for studying the biological functions of the bHLH transcription factor family of quinoa. In order to further study the evolutionary relationship of the CqbHLH transcription factor family, the phylogenetic tree of the family members was established using NJ method. The results showed that, as shown in Figure 2, CqbHLH transcription factor members could be divided into 21 branches, named A~U, and the number of branch members ranged from 2 (A and D) to 36(Q).

The 114 members of Quinoa GST gene family were identified by MEGA6.0. The maximum likelihood method was used to construct phylogenetic tree for 228 protein sequences from Quinoa, Arabidopsis and rice (Figure 3). The results showed that 114 members of Quinoa GST gene family were divided into seven groups, including GSTZ, GSTT, TCHQD. DHAR, GSTF, GSTL, GSTU. Among them, GSTU has 68 family members, accounting for 59.6%, which is quinoa. The largest branch of the GST gene family. GSTF, GSTL, GSTZ, GSTT, DHAR and TCHQD each contain 2. Three, six, six, five, four, two members of the family. They were named C for their classification and distribution on chromosomes, qGSTU1~68, CqGSTF1~23, CqGSTL1~6, CqGSTZ1~Z6, CqGSTT1~T5, CqDHAR1~4.

In general, the position of exons and introns can provide the most important information for the evolutionary relationship of species. The 114 gene structures of all identified quinoa GST gene families were analyzed, and the results were shown in (Figure 4). The number of exons contained in CqGST gene ranged from 1 to 14, among which CqGSTL2 contained 14 exons, indicating that CqGSTL2 contained 14 exons. The gene with the highest number of exons. Among the 68 CqGSTU members, except CqGSTU49, there are 9 exons. All the other Tau members contained 1-5 exons, of which 55 members contained 2 exons, accounting for 80.9% of the Tau group. Among the 6 CqGSTL members, CqGSTL2 contains 14 exons and CqGSTL1 contains 9 exons all other members contain eight exons. The number of exons in 6 CqGSTZ members was significantly different, including 3 members have 2 exons and 3 have 9 exons. CqDHAR2 was included in 5 CqDHAR members. It has 6 exons, and all the others have 5 exons. The five members of the CqGSTT class contain 6~9 exons. The two CQTCHQDS contain two and four exons respectively. 23 CqGSTF class members, except CqGSTF17. Except for 6 exons, the others all contained 1~3 exons, among which the most contained 3 exons, accounting for CqGSTF 60.9% of the class members. Genes in the same class often have similar structures, for example, 68 CqGSTU members CqGSTU49 contains 9 exons, and all other members contain 1~5 exons, including 55 exons. There are 2 exons, accounting for 80.9% of the Tau class. Similar to the gene structure, the conserved motif of quinoa GST protein was in the same class is usually composed of similar motifs. Similar gene structures and conserved motifs in each class are advanced Step proves the credibility of classification.

Studies on chromosome distribution of Quinoa showed in (Figure 5) that except for no CqGST gene on chr09, all the other chromosomes were identical there was CqGST gene distribution, and chr07 contains 20 CqGST genes, which was one of CqGST gene distribution. Hot spots. CqGSTL1 and CqGSTU1 cannot locate specific chromosomes due to the quality of the genome. Overall, there was no correlation between the number of GST genes on chromosomes and chromosome length.

The presence of two or more homologous genes in the 200 kb chromosome region was defined as tandem duplication. As shown in (Figure 6), there are 21 tandem repeats including 51 genes on 10 chromosomes of Quinoa. Accounting for 44.7%, referred to as tandem repeat gene cluster. Each of the 10 chromosomes contains 1-4 bases Chr16 has 4 GST gene clusters, which is the chromosome with the most GST gene clusters. The number chromosomes contain only one set of gene clusters (GST clusters). Each GST cluster contains 2, 4 genes, most of them The GST cluster has only two genes. Chr17 contains three gene clusters, one of which has four genes, was most genes GST cluster. In addition, a total of 51 genes formed 30 tandem duplicate gene pairs. The repeat event, MCScanX's method was used to obtain 27 duplicate gene pairs consisting of 51 genes. These results suggest that some CqGST genes may be produced by gene replication, while tandem and Fragment replication events may play a key role in the amplification of CqGST.

Transcription analysis by RNA sequencing (RNA-SEQ) is used to identify the tissue type-specific genes of interest mature tool. In order to explore the expression pattern of GST gene in Quinoa, different tissues of Quinoa or RNA-seq data from various developmental stages of organs were systematically analyzed. These samples were collected from the apical fractions. Biological tissue and other 11 tissues and organs (Figure 7). In class U, CqGSTU22, CqGSTU67, CqGSTU43, CqGSTU42, CqGSTU15; in class L, CqGSTL1 and CqGSTL5; in class Z, CqGSTZ5; DHAR class CqDHAR2 and CqDHAR3 were strongly expressed in various varieties, tissues and organs (Figure 8). And the CqGST, U66, CqGSTF20 and CqGSTF23 were not expressed in the 11 tissues or organs tested. It may be related to the temporal and spatial expression patterns of these genes. In addition, there were multiple genes in each type in the seedlings Medium and high expression, such as CqGSTL4, CqGSTL6.

4. Discussions

After the release of quinoa genome sequencing data, many gene families have been identified and verified at the whole genome level, including GRAS, ZIP ^{38, 39}. The second largest transcription factor family in plants, bHLH transcription factor family is involved in many pathways of plant growth and metabolism ⁴⁰. However, no such detailed study has been done on the quinoa bHLH family, and we identified 250 CqbHLH genes in quinoa. Based on the phylogenetic tree analysis, we divided these CqbHLHTFs into 21 clades, with 19 clades, the number of genes ranging from 3 (K) to 36 (D), and 2 clades A and D containing 2 genes. This is the same number of branches that have been reported for tomatoes ⁴¹, soybeans ⁴², and white pears ⁴⁴. However, these plants SlbHLHs (159) and MdbHLHs (188) have fewer bHLHs members than quinoa. This study was based on the published high-quality genome of Quinoa 114 members of Quinoa's GST gene family were identified, divided into seven major types, and like most other plants, GSTUs and GSTFs are the two largest, with 68 members and 23 members, respectively, while the other five divisions All have fewer than 7 members. The number of GST genes in Quinoa was much higher than that in Arabidopsis thaliana (66 GST genes) ⁴⁵, Rice (59 GST genes) ⁴⁶, poplar (81 GST genes) ⁴⁷, tomato (90 GST genes) ⁴⁸, soybean (101 GST genes) ⁴⁹, potato (42 GST genes) ⁵⁰ and other plants were advanced in the genome In the process of mutation, its size depends mainly on tandem and fragment replication, which is involved in the evolution of gene families. The number of CqGST genes in Quinoa has important implications, often leading to chromosome rearrangement and genomic instability amount is relatively high, which may be caused by species-specific fragment replication ^{51, 52}. In the course of evolution, gene replication plays an important role in the function of new genes and the expansion of gene families it plays a role ⁵³ and is associated with chromosome and morphological variation ⁵⁴. To better understand the CqGST gene family MCScanX's method was used to analyze the expansion mechanism of CqGST gene family. The analysis was obtained in total thirty tandem gene pairs formed by 51 genes and 27 fragment-replicating gene pairs formed by another 51 genes. These results suggest that both tandem and fragment replication events are major drivers of CqGST evolution. In addition, the synchro between Quinoa and its ancestral species C. allidicaule, C.suecicum, Arabidopsis and millet was constructed System comparison chart. It has been shown that quinoa is composed of C. allidicaule (A genomic diploid) and C.suecicum (B genome diploid) was hybridized about 3.3-6.3 million years ago ⁵⁵. There were several identical collinear gene pairs between C. allidicaule and C. Suggested that these genes had existed before quadrupling of quinoa while other genes may have evolved over time after the two species interbred or interbred to form quinoa In the process of gene replication and other forms. In addition, quinoa and Arabidopsis possible functional differences between CqGSTs under salt stress were analyzed using RNA-seq data from a public database The results showed that the expression level of CqGSTs gene was induced by salt stress, and part of Cq gene was induced by salt stress The expression of GST gene in both salt-tolerant and salt-sensitive quinoa cultivars was up-regulated with the extension of salt treatment time It is suggested that these genes play an important role in the response of quinoa to salt stress. Some studies have reported differences in the expression of GST genes in plants under abiotic stress, but only A few reports have confirmed their role in abiotic stress tolerance ^{56, 57}. Currently, it's used to study salinity most plants are CAM (crassulacean acid metabolism) plants, and this plant's genome is Unknown, quinoa has been sequenced as an ideal model for studying salt tolerance mechanisms in plants ⁵⁸. Therefore, this research Bioinformatics was used to analyze the transcriptase of two different Quinoa varieties under salt stress, and qRT-PC was used after verification, R selected eight CqGST genes in DHAR and GSTU subclasses. The expression of genes depends on the interaction between cis-acting and trans-acting elements can be analyzed by analyzing the cis-acting elements upstream of the promoter to predict the function of genes ⁵⁹. After the target genes were identified, the cis-elements in the promoter regions of these genes were identified and analyzed A piece. The results showed that these eight genes, in addition to having a large number of hormone-induced response elements and cis-regulatory elements, A large number of cis-acting elements related to plant stress response were also included, which was consistent with the experimental results of qRT-PCR above under salt and drought stress.

In the process of evolution, plants produce multiple members of a gene family mainly through extensive genome replication and diversification. WGD/ segment replication and tandem replication are the main causes of plant gene family expansion ⁶⁰. According to gene replication analysis, the main driving force of CqbHLH family expansion was genome replication and fragment replication, which was the same as that of apple ⁶¹. For example, using MCScanX (incomplete), 78% of the bHLH genes in quinoa were classified as WGD/ fragment copies, despite which 10% of the bHLH family members came by tandem. In addition, chromosome localization showed that CqbHLH genes were randomly distributed on 19 chromosomes of Quinoa, and there was evidence of fragment replication and tandem replication, which further indicated that these two replication modes played an important role in the expansion and evolution of the CqbHLH gene family in Quinoa. In addition, the Ka/Ks ratio suggests that CQBHLHS evolved mainly through purification selection, which may be due to selection to maintain important biological functions during their evolutionary history in order to adapt to the current environment and under the pressure of purification evolution. Exons and introns play key roles in plant evolution, and a large number of introns are lost over time in the early stages of species expansion ⁶². Thus number of introns increased through evolution, which may be a necessary mechanism for generating new gene functions. Intron-free members of the bHLH gene family in plants may have originated in prokaryotes and then replicated widely in plants. In this study, the structure of the bHLHTFs exon intron gene of quinoa showed that only 6.0% (15 genes) of CqbHLH TFs contained no intron and only one exon, which was basically consistent with the number of rice ⁶³. Therefore, we speculate that the bHLH gene family may be involved in many biological processes and molecular functions due to its evolutionary diversity and low conserved pattern. In addition, most bHLH proteins belonging to the same clave have similar motifs. Therefore, we speculate that members of the same branch may have similar functions. Numerous studies have shown that the bHLH transcription factor family plays an important regulatory role in plant abiotic stress response. Therefore, transcriptome data were used in this study to explore the expression patterns of CqbHLH gene family members in different tissues and the response of CqbHLH family members to salt stress. The bHLH transcription factor family was found to have significant tissue expression differences in Quinoa, suggesting that they play an important role in different development and physiological functions of quinoa. In addition, quinoa has been studied as a model for understanding plant salt tolerance, and bHLH genes identified in various plant species have been shown to play an important role in salt stress response. In addition, some CqbHLH transcription factors, such as CqXXX, were strongly expressed in Quinoa leaves according to the analysis results of the salt stress transcript data. After reviewing the literature, it was found that the CqXXX, gene did have salt tolerance function in other species. We speculated that these CqbHLHTFs might play an important role in regulating the formation of salt vesicles in Quinoa leaves and channling salt. This is consistent with previous reports. By comparing the expression differences of bHLHTFs between the two varieties before and after salt stress, we found that the expression levels of genes CqbHLH167, CqbHLH180, CqbHLH115 and CqbHLH215 in the sprouts of salt-tolerant varieties were significantly higher than those of sensitive varieties. To find the evidence of salt tolerance of the above differentially expressed genes in other species, this is the discussion, which cannot be limited to its own results.) Other genes, such as CqbHLH232, CqbHLH169, CqbHLH71, CqbHLH97, were significantly more expressed in the roots of salt-tolerant varieties than those of sensitive varieties. After stress treatment, its expression increased significantly. This suggests that these genes may be responsible for the differences in salt tolerance between the two varieties. In addition, in this study, some bHLH transcription factors, such as CqbHLH194, CqbHLH231, CqbHLH89 and CqbHLH62, were positively regulated by salt stress in both salt-tolerant varieties and salt-sensitive varieties of quinoa. These genes may not be the cause of salt stress resistance in salt-tolerant varieties and salt-sensitive varieties of Quinoa. The results of this study provide evidence for further identification of the functions of candidate genes in the process of plant salt stress resistance. In addition, some CQBHLHS were negatively responsive to salt stress, suggesting that they may be responding to other stresses or participating in other biological processes.

5. Conclusion

In this study, we identified 250 CqbHLHTFs from Quinoa and systematically analyzed their gene structure, conserved motifs, gene replication, evolutionary relationships and tissue expression patterns. According to phylogenetic analysis, the CqbHLH family was divided into 21 groups. According to collinearity analysis, WGD and tandem replication may play a role in the evolution of the CqbHLH family. In addition, according to RNA-seq data, CqbHLH gene may play an important role in coping with abiotic stress, and different varieties of Quinoa have different expression patterns under salt stress. (It was important to point out which genes are possible salt tolerance genes in the conclusion.). The data collected in this study provided the basis for further research on the bHLH gene and salt tolerance stress and other abiotic stress in quinoa, which will help to improve the economic value of quinoa.

Acknowledgements

This research was supported by the Research Program Sponsored by Ministerial and Provincial Co-Innovation Centre for Endemic Crops Production with High-quality and Efficiency in Loess Plateau, Taigu 030801, China (No. SBGJXTZX-15), the National Modern Agricultural Industrial Technology System (CARS-03-01-24), the Key Laboratory of Crop Ecology and Dryland Cultivation Physiology of Shanxi Province (201705D111007), the Key Innovation Team of the 1331 Project of Shanxi Province, the Scientific and Technological Innovation Project of colleges in Shanxi Province (2021L178), Science and technology innovation fund of Shanxi Agricultural University (2018YJ18).

References