Cloning And Analysis Of KcHTRs Gene Family From Desert Plant Karelinia Caspia

Plants always encounter many non Biotic stress in the whole process of their growth cycle.In recent years,more and more studies have been conducted on plant non Biotic stress,of which 14-3-3 protein is one of the more widely studied members.In the earlier stage,we found 13 14-3-3 genes in this species from the high-temperature normal temperature transcriptome data of Karelinia caspia,of which 7 genes have obvious expression differences.Through online comparison with NCBI,we found that they belong to the 14-3-3 gene family,which is named KcHTRs gene family in this study.Since 14-3-3 protein is mainly involved in plant responses to salt,drought and low temperature stress in plants,few reports have reported that it is involved in response to high temperature stress.However,our research team found that the gene family has obvious differential expression in the transcriptome data of early high temperature treatment,so this study conducted research on the possible extreme temperature tolerance of the KcHTRs gene family of Karelinia caspia.The research contents and results are as follows:（1）Cloning and Expression Pattern Analysis of KcHTRs gene family from Desert Plant Karelinia caspia:Seven members of the KcHTRs gene family were successfully cloned.Sequencing revealed that the total length of KcHTRs gene family CDS was 708bp～789bp,encoding 235～262 amino acids.The sequence consistency of the KcHTR1 with the 14-3-3 protein of Smallanthus sonchifolius and Helianthus annuus in the Asteraceae Bercht family was 97.36%;The sequence consistency between KcHTR2 and the 14-3-3protein of Cynara scolymus in Asteraceae Bercht was as high as 96.90%;The protein sequence of KcHTR3was 97.68%consistent with the 14-3-3 protein of Erigeron canadensis Asteraceae Bercht;The sequence consistency between KcHTR4 and the 14-3-3 protein of Cynara scolymus in Asteraceae Bercht was as high as 99.61%;The protein sequence of KcHTR5 and the 14-3-3 protein of Smallanthus sonchifolius Asteraceae Bercht had a high consistency of 95.04%;The protein sequence of KcHTR7 and the 14-3-3 protein of Smallanthus sonchifolius Asteraceae Bercht had a high consistency of 98.84%;KcHTR8 and the 14-3-3protein sequence of Smallanthus sonchifolius have a high consistency of 97.23%.The corresponding bioinformatics analysis and phylogenetic analysis were conducted for these 7 members,and it was found that the 14-3-3 protein encoded by KcHTRs is highly conserved.Through the construction of a phylogenetic tree and online NCBI comparison,it was found that the KcHTRs gene family of Karelinia caspia has a high degree of homology with Smallanthus sonchifolius and Helianthus annuus in the Compositae family.Analysis of various abiotic stress expression patterns showed that KcHTR1 had good resistance to extreme temperature and high osmotic stress;KcHTR2 mainly has resistance to extreme temperatures;KcHTR3mainly has good resistance to extreme temperatures and drought;KcHTR4 has good resistance to extreme temperatures and salt;KcHTR5 mainly responds to high temperature and drought stresses;KcHTR7 has resistance to extreme temperatures and drought;KcHTR8 has resistance to extreme temperatures and drought.（2）Functional verification of desert plant Fagaceae KcHTRs gene family in bacteria:By constructing prokaryotic expression vector of KcHTRs and transforming it into Escherichia coli BL21,it was determined that the optimal induction system of KcHTRs expressed proteins was 37℃,OD₆₀₀ reached 0.8,the optimal induction concentration of IPTG was 0.5 mmol/L,and the induction time was 8 h.SDS-PAGE analysis showed that the protein sizes expressed by the above 7 genes ranged from 27 k Da to 29 k Da,which was consistent with the gene size.Through the stress resistance analysis of the transgenic Escherichia coli BL21,it was found that KcHTRs could significantly improve the tolerance of host bacteria to 45℃high temperature,4℃low temperature and 0.2 mol/L Na Cl.（3）KcHTR3 and KcHTR4 genes of desert plant Karelinia caspia Functional analysis of stress resistance:Through the successful construction of KcHTRs plant expression vector,we successfully obtained the pure line plants of overexpression KcHTR3 and KcHTR4 generation T3 of Arabidopsis and transgenic plants of cotton generation T0.Through the determination of physiological and biochemical indicators of overexpression Arabidopsis,the results showed that KcHTR3 and KcHTR4 can significantly improve the ability of Arabidopsis to withstand 40℃and 4℃low temperature,thus determining that KcHTR3 and KcHTR4 have the function of responding to extreme temperature.This study cloned 7 KcHTRs genes and found through homology comparison that these genes belong to the 14-3-3 family;Through prokaryotic expression,it is found that the gene family can express corresponding proteins,and can improve the tolerance of E.coli to high and low temperatures.Some genes also have salt tolerance and drought tolerance;Among them,KcHTR3 and KcHTR4 transgenic E.coli had more significant tolerance to high and low temperatures.At the same time,by overexpressing KcHTR3 and KcHTR4 genes in Arabidopsis thaliana,it was found that transgenic Arabidopsis thaliana had significantly improved tolerance to extreme temperatures.This study provides a research basis for clarifying the specific stress resistance functions of the KcHTRs family in Karelinia caspia,and for the subsequent application of stress resistance such as KcHTRs in molecular breeding of related crops such as Gossypium spp.