Promoter Analysis Of Ver2 Gene And Its Regulation Of Chloroplast Gene Expression In Wheat Albinism Line

The albinism phenomenon of wheat albinism line is related to the interaction of nuclear and cytoplasm,which is controlled by nuclear and cytoplasm genes.Ver2 gene is one of the candidate gene to albinism,which is related to the vernalization of wheat and special in wheat.The Ver2 gene encodes a lectin protein,which induced by low temperature.Our preliminary study showed that the sequence of the Ver2 gene coding region in the wheat albinism line was consistent with the reported sequence,but the sequence of 1611 bp was absent in the promoter region.Ver2 gene in the wheat albinism line was expressed at room temperature and only in the interior leaf.In this study,we focused on the study of the function of the promoter about Ver2 gene and the Ver2 gene on the regulation of plastid gene expression,hoping to laid a foundation for revealing the molecular mechanism of albinism phenomenon.In this study,the distribution of Ver2 gene and its promoter in 47 cultivated wheats was identified.And Ver2 gene was edited and overexpressed by transformation.The transient expression system was used to detect the activity of Ver2 gene promoter.Futhermore,in order to investigate whether the Ver2 gene regulates the expression of chloroplast gene and the development of chloroplast,we genetic transformed the Ver2 gene promoter in Arabidopsis thaliana.Last,the immunoprecipitation technique was used to identificed VER2 binding protein in transgenic plants.The main results are as follows:(1)The Ver2 gene was identified from 47 different cultivated wheats,and the result showed that Ver2 gene was amplified from 35 cultivars and there was no specificity in geographical distribution and germplasm.The genetic evolution analysis showed that VER2 protein was closest to the mannose/glucose-specific lectin in Aegilops tauschii.The promoter region of Ver2 gene in wheat had at least three variation types: one was about 5900 bp containing three small repeats and two large repeats.The second was deletion of 1611 bp in the gene upstream and having complete size of the big and small repeats.The third is the promoter which the sequence information is not clear.(2)The study on Ver2 gene overexpression line VF3 showed that the overexpression of Ver2 gene had the effect of promoting albinism.Five expression vectors of gRNA/Cas9 were constructed according to CRISPR/Cas9 technique,which laid a foundation for the study of Ver2 gene editing.The p35S::Ver2 gene was transferred into the wheat without Ver2 gene and the T1 transgenic plants were obtained.(3)Small repeat(PVS)and large repeat(PVL)of the Ver2 gene promoter were ligated to the expression vector PBI121,and were transiently transformed in Arabidopsis thaliana and tobacco.GUS staining indicated that PVS and PVL could induce the expression of gus gene and the transcriptional activity of the promoter was established.The expression vectors of Ver2 gene was constructed by using pCAMBIA1301 as the basic framework and PVS,PVL and 2kb as the promoter region,then those vectors were transformed in Arabidopsis thaliana and wheat and the T1 transgenic plants were obtained.(4)Compared with the control Arabidopsis,the flowering in advance in transgenic p35S::Ver2 plants,revealing exogenous Ver2 gene can promote plant flowering.The expression of 11 chloroplast coding genes at different temperature conditions was detected by qRT-PCR.The results showed that the expression of 11 chloroplast coding genes in wild plants was significantly higher than that in Transgenic plants at room temperature and 4℃ for20 days,and up-regulated at 4℃ for 40 days.In conclusion,Ver2 gene was widely found in wheat.There were at least three variants of Ver2 gene promoter in wheat and the large repeat and small repeat has promoter activity.The Ver2 gene can promote albino in wheat,promote the flowering and regulate the expression of chloroplast gene in Arabidopsis thaliana.The the function of Ver2 gene and its mechanism in the albino phenomenon is still to be further studied.