Expression Analysis Of TaNAC14 And TaRZ2 In Vitro And Identification Of Wheat Overexpressing-Transgenic Lines

Wheat is one of the most important food crops in the world,and its production is directly related to world food security;therefore,high-yield quality breeding has always been the main target of wheat breeding.The functional research of wheat growth and development and stress related genes in stress response can provide theoretical basis and scientific basis for high-yield wheat breeding.In the early stage of the research,we obtained the target genes TaNAC14（NAC14 gene in wheat）and TaRZ2（zinc finger Glycine-rich protein 2）of the high-expression miR164 in the seedling and grain period and miR9655 specifically express during the wheat grain period.The biological role of these target genes in wheat growth and development is still unclear.In order to study the biological functions of TaNAC14 and TaRZ2 genes in wheat growth and development,this study firstly used bioinformatics methods to analyze the physicochemical properties and structure of TaNAC14 and TaRZ2 gene-encoding proteins.Secondly,the prokaryotic expression vector of TaNAC14 and TaRZ2 were constructed respectively.The expression vectors pET21a-TaNAC14 and pGEX-6p-1-TaRZ2 were expressed and purified in vitro by E.coli expression system.The nucleic acid binding activity of TaRZ2 protein was verified in vitro.In addition,in this study,the combination of BASTA smearing and PCR detection was used to identify the transgenic plants of TaNAC14 and TaRZ2 transgenic wheat,which were created by Agrobacterium-mediated transformation.The copy number of T₀ transgenic wheat was detected by TaqMan real-time PCR.At the same time,the genetic stability of T_0:1 transgenic wheat lines was analyzed,and the expression level of target genes of T₁ transgenic lines was determined.The main findings of this study are as follows:1.This study obtained TaNAC14 protein expressed in large amounts in E.coli.The protein is mainly present in a soluble form in the supernatant,and is purified and purified by Ni gel column to obtain a relatively pure target protein.2.Nine positive plants were identified from 14 T₀ generation TaNAC14 transgenic plants;5 of them were single copy plants.Analysis of the genetics of the T_0:1 lineage target genes of these single copy transgenic plants indicated that TaNAC14 is heritable.The expression level of TaNAC14,a target gene of T₁ transgenic wheat lines,was determined.The results showed that compared with wild type,the expression level of target genes of T₁ generation TaNAC14transgenic wheat lines increased by 16-95 times.3.This study obtained TaRZ2 protein expressed in E.coli,which was mainly present in soluble form in the supernatant;it was isolated and purified by GST gel column to obtain pure protein of interest.4.Nucleic acid binding experiments in vitro on purified TaRZ2 protein showed that the protein has nucleic acid binding activity and binds to both ssDNA and dsDNA in vitro.5.Twenty-two positive plants were identified from 39 T₀ generation TaRZ2 transgenic regenerated plants,8 of which were single-copy transgenic plants.Analysis of the genetics of the T_0:1 strain target genes of these single-copy transgenic plants indicated that TaRZ2 is heritable.The expression level of the target gene TaRZ2 of T₁ transgenic wheat lines was determined.The results showed that compared with the control JW1,the expression level of the target gene of TaRZ2 transgenic wheat lines increased by 0.4 to 26 times.In summary,the expressed proteins of TaNAC14 and TaRZ2 were obtained by prokaryotic expression system in vitro and prokaryotic expression protein isolation and purification techniques,and the nucleic acid binding activity of purified TaRZ2 protein was verified in vitro.In addition,the identification of TaNAC14 and TaRZ2 transgenic wheat yielded a single copy of TaNAC14 and TaRZ2 transgenic wheat lines that are stably inherited and can be expressed at high levels.These studies laid the foundation for the subsequent functional studies of TaNAC14 and TaRZ2,providing new genetically stable germplasm materials for wheat breeding.