Functional Analysis Of Potato StMYB2R-86 Gene Under Salt Stress And Identification Of StHsp20 Gene Family

Soil salinization is one of the important ecological and environmental problems in current agricultural production.Salt stress seriously affects the germination and growth of crop seeds,resulting in a decline in crop yield and quality.In order to cope with unfavorable environmental conditions,plants have formed a series of complex regulatory mechanisms during evolution.The transcriptional regulatory network which relies on transcription factors plays an important role in plant growth and development and resistance to stress and other physiological processes.MYB transcription factor family is a large transcription factor family in plants,and many researches about their roles in plant stress response have been reported.There are very few reports on the function of MYB transcription factor in response to stress in potato.In order to study the function of potato MYB transcription factor under salt stress,this study identified potato 2R-MYB genes on genome-wide level,and screened out potato 2R-MYB genes related to abiotic stress response.In the study,the St MYB2R-86 gene was cloned to further analyze its function in plant salt stress response.During the process of research,it was found that the expression leveles of Hsp20 genes which code heat shock proteins were significantly up-regulated under salt stress,therefore,potato Hsp20 genes were also identified and analyzed their function.The main findings are as follows:（1）110 potato 2R-MYB genes were identified,which are distributed on 12 potato chromosomes and named as St MYB2R-1 to St MYB2R-110 according to their chromosomal locations.Through the analysis of RNA-seq data,53 potato 2R-MYB genes that respond to salt stress,drought stress or high temperature stress were screened out,and the expression leveles of several 2R-MYB genes were detected by real-time fluorescent quantitative PCR.The q RT-PCR results were consistent with the results of RNA-seq analysis.（2）The potato St MYB2R-86 gene was cloned,and two red fluorescent protein fusion expression vectors St MYB2R-86::RFP and RFP::St MYB2R-86 were constructed,both of which indicated that St MYB2R-86 protein was a nuclear-localized protein.The expression level of St MYB2R-86 gene under salt stress,drought stress and high temperature stress were detected,and the results showed that St MYB2R-86 gene responded to the three kinds of abiotic stresses.（3）Potato St MYB2R-86 gene was heterologously expressed in Arabidopsis.Under normal growth conditions,there was no significant difference between wild-type Arabidopsis and St MYB2R-86 overexpressed Arabidopsis.Under 100 m M Na Cl salt stress,the germination speed and germination rate of St MYB2R-86 gene overexpressed Arabidopsis seeds were both higher than those of wild-type Arabidopsis,and the root length（P<0.01）and fresh weight（P<0.05）of transgenic Arabidopsis were significantly higher than those of wild-type Arabidopsis.Under salt stress,expression levels of stress-related genes At NHX1,At PERX34,At SOD,At P5CS2,At LEA3,At HSP17.8 and At HSP17.6B in St MYB2R-86 gene overexpressed Arabidopsis were significantly higher than those of wild-type Arabidopsis.No significant difference of ascorbic acid peroxidation enzyme gene At APX1 expression level was detected between St MYB2R-86 gene overexpressed Arabidopsis and wild-type Arabidopsis.In addition,the up-regulation of At HSP17.8 and At HSP17.6B genes was higher than that of the other genes,and the expression level of At HSP17.8 in St MYB2R-86 gene overexpressed Arabidopsis was significantly higher than that in wild-type Arabidopsis without salt stress treatment,which implying the potential function of Hsp20 gene family in plant salt stress resistance.（4）Under normal growth condition,no significant differences were observed between the phenotypes of wild-type potato and St MYB2R-86 gene over-expressed potato.After growing for 1 month under 100 m M Na Cl salt stress,St MYB2R-86 gene overexpressed potato grew stronger than wild-type potato.The plant height was significantly higher（P<0.05）than wild-type potatoes,and the root system was relatively more developed.Physiological indicators showed that St MYB2R-86 gene overexpressed potato accumulated more proline under salt stress,cell ion leakage and malondialdehyde content were lower than those of wild-type potato,and the distribution of superoxide O^2-in leaves was also less than that of wild-type potato.（5）The analysis of cis-acting elements in the potential promoter region located at2000 bp upstream of the coding region of potato St MYB2R-86 gene showed that the region contained four hormone-responsive cis-acting elements,which respond to methyl jasmonate,abscisic acid and salicylic acid,respectively.Hormone treatment was applied on wild-type Arabidopsis and St MYB2R-86 gene overexpressed Arabidopsis.Under the treatment of abscisic acid and salicylic acid,there were no significant differences on the phenotype of wild-type Arabidopsis and St MYB2R-86 gene overexpressed Arabidopsis.Under the treatment of methyl jasmonate,the root length and fresh weight of St MYB2R-86gene overexpressed Arabidopsis were significantly higher（P<0.01）than that of wild-type Arabidopsis.Expression levels of jasmonic acid signaling pathway related genes in St MYB2R-86 gene overexpressed Arabidopsis showed that with the continue of salt stress,the expression levels of At JAR1,At COI1 and At MYC2 genes were continuously down-regulated,and expression level of the negative regulator of the jasmonic acid signaling pathway,At JAZ1 gene,were gradually increased.Wild-type Arabidopsis showed opposite results of gene expression level,indicating that potato St MYB2R-86 gene may negatively regulate the jasmonic acid signaling pathway to enhance the plant salt stress tolerance.（6）Forty-eight potato Hsp20 genes were identified,which were distributed on 12potato chromosomes.Among the 48 potato Hsp20 genes,19 genes were tandem duplicated genes,and 2 genes were segmentally duplicated genes,indicating that gene duplication events may be crucial to the expansion of potato Hsp20 gene family.Forty-three potato Hsp20 genes contained no intron or only one intron,which may be related to quick response under stress.The analysis of cis-acting elements showed that,except for St Hsp20-23 and St Hsp20-41 genes,the promoters of the other potato Hsp20 genes contained at least one cis-acting element related to stress response.The expression level changes of 48 potato Hsp20 genes under high temperature,salt and drought stress were detected by fluorescence real-time quantitative PCR.The results showed that expression levels of most Hsp20 genes responded to the three abiotic stresses,indicating that potato Hsp20 genes may be vital in abiotic stress response.