Analysis Of IQD Gene Family And Functional Identification Of IQD5 And IQD35 For Drought Tolerance In Brassica Rapa

Brassica rapa crops mainly contain Chinese cabbage(Brassica rapa ssp.pekinensis)and Non-heading Chinese cabbage(Brassica rapa ssp.chinensis),which belong to the cruciferae family of Brassica plants.Chinese cabbage and Non-heading Chinese cabbage are rich in vitamins,cellulose,carbohydrates and other nutrients,and are one of the best-selling vegetables in China.Drought stress is the agricultural meteorological disaster that affects the planting area,yield and quality of Brassica rapa.Related studies have shown that the IQD(IQ67 domain-containing protein)gene family plays an important role in the development of a variety of plants,while most of IQDs are responsive to drought stress.Based on the published Chinese cabbage genome,we performed a systematic genome-wide analysis of IQD gene family in Chinese cabbage and analyzed their response to drought stress.On this basis,the BrIQD5 and BrIQD35 genes,which are involved in drought stress,were functionally analyzed and identified by transgenic,yeast two-hybrid,bimolecular fluorescence complementation(BiFC),cDNA fragment truncation and mutation techniques.BcIQD5,a Non-heading Chinese cabbage gene,is the homologous gene of BrIQD5,so we have also analyzed and identified the structural features and functions of BcIQD5 in this study.The discovery helps to elucidate the molecular mechanism of the IQD gene family involved in plant drought resistance,which will be of great significance for the genetic improvement of Brassica rapa.The results are as follows:1 Genome-wide identification and expression analysis of the IQD gene family in Chinese cabbageA total of 35 IQD genes were identified in Chinese cabbage by Blastp search and verification,named BrIQD1?BrIQD35,and they can be divided into three subfamilies according to phylogenetic analysis.The same subfamily has similar intron-exon distribution and conservative motif components.35 BrIQDs are distributed unevenly on 9 of the 10 chromosomes,with 4 segmental duplication gene pairs.The BrIQD gene family had tissue-specific expression patterns and 32 of them responded to drought stress.The expression levels of BrIQD5 gene was significantly up-regulated under drought stress,and the expression pattern of BrIQD35 gene under drought stress also changed significantly.2 IQD5 from Chinese cabbage and Non-heading Chinese cabbage can enhance the drought tolerance of plantsIn order to verify the role of Brassica rapa IOD5 in plant drought response,the Chinese cabbage IQD5(BrIQDS)and Non-heading Chinese cabbage IQD5(BcIQD5)overexpressed vectors were constructed,and transgenic tobacco and Arabidopsis plants were obtained by genetic transformation.At the same time,the knockdown of BrIQD5 was carried out by virus-induced gene silencing method.The results of drought stress treatment showed that the BrIQD5-overexpressed plants were more tolerant to drought stress than wild-type plants,while the BrIQD5-silenced plants reduced drought tolerance.BcIOD5-overexpressed plants showed stronger drought tolerance than wild type plants,indicating that IQD5 from Brassica rapa was an important component of plant drought response.3 Screening and verification of IQD5 interacting proteins in Chinese cabbage and Non-heading Chinese cabbageIn order to elucidate the mechanism of IOD5 enhancing plant drought tolerance,BrIQD5 was used as a bait protein to screen for the interacting proteins by yeast two-hybrid library screening system.A total of six stress-related proteins were obtained,including two calmodulin proteins(CaMa and CaMb)and four drought-related proteins,these six genes were all responded to drought stress by qRT-PCR technique.In view of the important role of CaMs in many physiological and biochemical processes of plants,we further validated the interaction of IQD5-CaMa and IQD5-CaMb using yeast two-hybrid,BiFC technology and cDNA fragment truncation techniques.BrIQD5 contains two IQ motifs,IQ₁ is the binding site of CaMa and CaMb,and isoleucine is an essential amino acid for BrIQD5-BrCaMa and BrIQD5-BrCaMb interaction.BcIQD5 contains three IQ motifs,IQ₁ and IQ3 are binding sites of CaMa,IQ₁,IQ and IQ3 are binding sites of CaMb.4 Functional analysis of IQD35 involved in drought stress in Chinese cabbageIn order to verify the role of BrIQD35 gene in plant drought response,a BrIQD35-overexpressed vector was constructed and transgenic tobacco plants were obtained through genetic transformation.The results of drought stress-treatment indicated that BrIQD35 was localized on the plasma membrane and overexpression of BrIQD35 can enhance the drought tolerance of plants.The domain analysis of BrIQD35 showed that it contained an IQ motif(calmodulin binding site).The results of yeast two-hybrid assay showed that BrIQD35 had a weak interaction with BrCaMb,but did not interact with BrCaMa,suggesting that BrIQD35 might function through the Ca²⁺_CaMb pathway.In summary,IQD5 and IQD35 in Brassica rapa may enhance the drought tolerance of plants through the Ca²⁺_CaM pathway.Exploring the function of the IQD5 and IQD35 is important for improving the quality and yield of Brassica rapa.