Identification And Function Analysis Of The RNase T2 Gene Family In Chinese Jujube(Ziziphus Jujuba Mill.)

Chinese jujube(Ziziphus jujuba Mill.)is native to China and widely cultivated,which is an important dry fruit tree species and economic forest species that can be used for medicine and edible.Ribonuclease(RNases),a kind of nucleic acid hydrolase,is widely existing in animals and plants and can be divided into three types:RNase T1,RNase T2 and RNaseA.Previous studies have shown that RNase T2 family is involved in the process of stress resistance,senescence and abscission,self incompatibility,but there are few studies on RNase T2 family gene of Chinese jujube now.Chinese jujube genome sequencing completed in 2014 might be a base to reveal the role of RNase T2 gene family at the genomic level.In this study,the RNase T2 family genes of Chinese jujube were identified using HMM and then analyzed by the bioinformatics software such as ExPASy,SignaIP,GSDS and MEGA7.Real-time quantitative RT-PCR(qRT-PCR)and transgenic Arabidopsis experiments were carried out to explore the function of these genes.The main results of this study were as follows:1.Five RNase T2 genes were obtained in Chinese jujube genome,encoding five acidic,unstable and hydrophobic proteins.Among the 5 proteins,ZjRNase2 and ZjRNase3 were highly similar,ZjRnasel-ZjRnase4 were secretory proteins,and only ZjRNase5 did not contain signal peptide.The homologous sequence analysis of RNase T2 family proteins showed that the proteins of Chinese jujube were similar to those of apple,peach,pear,orange,grape and Arabidopsis.The molecular weight of ZjRNase5 protein was far beyond the range of RNase T2 gene family,and it also contained the conservative structure of PLN00113 family.So,ZjRNase5 classification was not clear.2.ZjRNase1 gene contained two introns and its cDNA total length was 681bp.ZjRNase1 protein contained 226 amino acids and had a highly similar conserved domain to class Ⅰ protein in tomato,Arabidopsis and rice.Except for a large number of elements related to abiotic stress,there were also cis acting elements belonging to plant tissue-specific promoters in ZjRNase1 gene.The qRT-PCR analysis of gene expression showed that Chinese jujube ZjRNase1 gene expression was strong tissue-specific,which was much higher in stems,leaves and flowers than in fruits and roots.According to phylogenetic tree analysis,the gene might belong to class Ⅰ.In Arabidopsis,it was found that the leaves of transgenic plants curled after overexpression ZjRNase1 gene.It indicated that the gene involved in leaf development and might play a role in plant self-defense.3.ZjRNase2 gene contained 7 introns,with the full cDNA length of 843bp.ZjRNase2 protein contained 280 amino acids,with conserved domain different from the class Ⅲprotein(S protein)of apple,pear and almond,but a highly similar conserved structure to S-like RNases protein of apple,pear and Arabidopsis.Cis element prediction showed that the promoter of ZjRNase2 gene might be inducible.The results of expression analysis showed that it was highly expressed at the small bud stage of flower development,and the full red stage of fruits.According to phylogenetic tree analysis,the gene might belong to class Ⅱ.Over-expression of ZjRNase2 gene in Arabidopsis exhibited that the pods of transgenic plants were shorter,shrunk or short of seeds compared with wild-type plants.These indicated that ZjRNase2 gene played a role in the development of fruits and seeds,and its overexpression might result in stunted development of pods in transgenic Arabidopsis.4.ZjRNase4 gene contained one intron and the full length of its cDNA was 729bp.ZjRNase4 protein included 242 amino acids and its conserved domain was different from that in the class Ⅱ protein(S-like RNases protein)of apple,pear and Arabidopsis,but similar to that in class Ⅰ and class Ⅲ protein(S protein)of other species.The prediction results of cis elements in the promoter of ZjRNase4 gene were consistent with ZjRNasel.The expression of ZjRNase4 gene was found to be high in the flowers.The expression in different stages of flower showed that it was higher expression in mature buds than other stages.More according to phylogenetic tree analysis,the gene should belong to class Ⅲ.But the isoelectric point of ZjRNase4 showed acidity.Although it had similar conservative structure with other species,its amino acid sequence was quite different.So,it might be a special S gene.The analysis of the full-length cDNA sequence of ZjRNase4 showed that it had the basic characteristics of S-RNase gene.The gene was a nuclear and membrane protein,which was proved by subcellular localization.Yeast two hybrid test showed that there were no interaction between ZjRNase4 protein and Skp1,and no transgenic Arabidopsis positive vaccine with ZjRNase4 gene was screened,so the function of this gene still needs to be further explored.In conclusion,five genes of RNase T2 gene family were obtained from the whole genome data of Chinese jujube.Based on bioinformatics analysis,real-time gene expression analysis,transgenic analysis of Arabidopsis,subcellular localization and yeast two hybrid test,the functions of ZjRnase1,ZjRnase2 and ZjRnase4 were preliminarily revealed.This study provided a basis for the further study on the function and mechanism of RNase T2 family gene and promoting its application in practice.