Functional Analysis Of KcVP1 Gene In Karelinia Caspia For Plant Stress Resistance And Early Flowering

The Tarim Basin,with its complex natural conditions of perennial drought and low rainfall,wind and sand,and extreme temperatures,has produced special organisms that can adapt to a variety of adversity environments.Our group found that the overexpression of the Na~+/H~+reverse transport protein gene in Arabidopsis thaliana can not only improve the salt and drought tolerance of Arabidopsis thaliana,but also have the characteristics of high-temperature tolerance,and also promote the early flowering phenomenon of transgenic Arabidopsis thaliana.In this thesis,we analyzed whether the inorganic pyrophosphatase(H~+-PPase)gene of the vesicular membrane of Karelinia caspia also has the function of stress tolerance and promoting early flowering in plants by cloning it,and provided a research basis of genetic resources for molecu Lar breeding of stress tolerance and early flowering in crops.The results of the study are as follows:Cloning and bioinformatics analysis of KcVP1 gene in desert plant Flos Daturae.The KcVP1 gene was successfully cloned and sequenced to find that its c DNA is 2286 bp in length,encoding amino acids 761amino acids,with a molecular weight of 80.32 k Da and a theoretical p I value of 5.03.It is a transmembrane protein containing 13 transmembrane structures,and its similarity value with the VP1 protein of sunflower is 53%,so it is named KcVP1.Functional analysis of the KcVP1 gene in E.coli and Saccharomyces cerevisiae for resistance.KcVP1 was constructed into a prokaryotic expression vector and yeast expression vector and transferred into E.coli BL21 and yeast strain Y2HGold,respectively.By analyzing the reproduction rate of the transformed strains under different adversity treatments,it was found that the reproduction rate of E.coli and yeast transformed with the KcVP1 gene was significantly higher than that of the control strain under high salt,drought,and high-temperature conditions,indicating that The results showed that the reproduction rate of E.coli and yeast transformed with KcVP1 gene was significantly higher than that of the control strain under high salt,drought,and high-temperature conditions.KcVP1 gene enhances the resistance of transgenic Arabidopsis thaliana seeds during germination.The gene was overexpressed in Arabidopsis thaliana and treated with 100 mmol/L Na Cl and 20%PEG 6000stress during germination of T3-generation Arabidopsis seeds,and the results showed that the germination rate of transgenic Arabidopsis thaliana was about 1.5 and 1.3 times higher than that of wild-type Arabidopsis thaliana,respectively,indicating that the gene has excellent salt and drought tolerance.The Kc NHX1 and KcVP1 genes of Karelinia caspia have the function of promoting early flowering in Arabidopsis thaliana.By counting the reproductive developmental indexes of transgenic and wild-type Arabidopsis thaliana,it was found that the time of moss extraction and flowering of Arabidopsis thaliana transgenic to Kc NHX1 and KcVP1 genes was 7-9 d earlier and seed maturation was 12-15 days earlier relative to wild-type Arabidopsis thaliana.By further analyzing the endogenous hormones IAA,GA,and BR in Arabidopsis thaliana Kc NHX1 and KcVP1 gene overexpression strains and wild-type Arabidopsis thaliana plants,it was found that CTK,and BR,it was found that the endogenous hormone content of Arabidopsis thaliana transgenic to Kc NHX1 and KcVP1 genes was higher than that of wild type,indicating that the Karelinia caspia Kc NHX1 and KcVP1 genes have the function of promoting early flowering in plants,and this function may be related to the accumulation of IAA,GA,CTK,and BR hormones.Expression pattern analysis of regulatory genes related to the plant flowering pathway revealed that SOCI and AGL24 expression of Kc NHX1 and KcVP1 gene overexpression strains promoted early flowering in Arabidopsis.CONCLUSION:In this thesis,by cloning the KcVP1 gene from the desert plant Flos Daturae and performing preliminary validation of its function in Escherichia coli,yeast,and Arabidopsis,we found that the KcVP1 gene from Flos Daturae not only has a high temperature,salt and drought tolerance but also both KcVP1 and Kc NHX1 genes from Flos Daturae have the effect of promoting early flowering in plants,which is important for selecting and breeding resistant and early maturing crop varieties such as cotton This is important for the selection of resistant and early-maturing varieties of cotton and other crops.