Cloning And Characteristic Of Jasmonate-induced Protein Gene(TwRIP1) From Triticale(X Triticosecale Wittmack)

Objective: drought is one of the main abiotic stress factors affecting crop quality and yield.Plants will accumulate a large amount of jasmonates under stress.Jasmonates activate the expression of specific genes through signal transduction,thereby synthesizing large amounts of jasmonate-induced proteins which are involved in plant growth and development and resistance to biotic and abiotic stress.To understand the molecular mechanism of triticales to resist drought stress and acquire efficient genes which are very useful to improve plant abiotic stresses tolerance,in this study,a jasmonate-induced gene（TwRIP1）which was up-regulated by drought stress significantly was cloned from hexaploid triticale.Methods: a full-length of TwRIP1 gene coding region（CDS）which was up-regulated significantly under drought stress was cloned from hexaploid triticale by using RT-PCR method,which was based on early RNA-seq transcriptome sequencing data from triticale.And the bioinformatics of the gene was analyzed with online software.The relative expression level of TwRIP1 induced by drought stress,20%PEG 6000,200 mM NaCl,4 ℃ low temperature stress,100 μM MeJA and 100 μM ABA treatment in organs of triticale was analyzed using qRT-PCR technology.The TwRIP1 in triticale was silenced using the BSMV-VIGS technique.And the pattern of gene expression and drought resistance physiological indexes under drought stress and with 100 μM MeJA treatment were tested.The over-expression vector with the target gene was constructed and Arabidopsis thaliana was infected with dip-flower method and transformed wheat with immature embryo using agrobacterium-mediated method.Results: a full-length 852 bp of jasmonate-induced gene TwRIP1 CDS（GenBank ID: MT159334）encoding 283 amino acids was cloned from hexaploid triticale.The predicted molecular weight of the TwRIP1 protein was 30 kDa,and there was no signal peptide and transmembrane protein.It was predicted that TwRIP1 protein had 10 Ser phosphorylation,16 Thr phosphorylation and 5 Tys phosphorylation sites,and TwRIP1 was located in the cytoplasm.TwRIP1 had 98.23 % homology with TRIDC6BG073370 and TRIUR3₀9425.The protein sequence between 10 ～ 211 amino acids of TwRIP1 is Ribosome inactivatingprotein（RIP）conservative functional domains,belong to RIP（PF00161）protein family.The RIP conserved structure domain of TwRIP1 contained five amino acid residues constituting the active site of rRNA N-glycosidase.There were differences in the expression levels of TwRIP1 gene in different tissues of triticale,with the highest expression in leaves,the second in roots,and the lower in stems and grains.The expression of TwRIP1 gene was significantly up-regulated under drought stress,20% PEG6000,200 mM NaCl,100 μM ABA,100 μM MeJA and 4 ℃ low temperature.Among them,the leaves of triticale were very sensitive to the reactions of 20% PEG6000 and 100 μM MeJA,while the roots of triticale were more sensitive to the reactions of 100 μM ABA.In addition,the expression of TwRIP1 gene in roots increased more than that in leaves under abiotic stress and hormone treatment.The expression level of TwRIP1 in triticale inoculated with BSMV: TwRIP1 decreased significantly compared with that in the control.Compared with WT and BSMV: 00,The proline content and SOD activity of TwRIP1 gene silencing in triticale significantly decreased under drought stress and MeJA treatment.The relative water content andnet photosynthetic rate of TwRIP1 gene silencing in triticale leaves also decreased significantly under drought stress.The over-expression vector of TwRIP1 gene,pWMB201-ubi-TwRIP1,was constructed and three positive transgenic wheat plants were obtained.However,many transgenic Arabidopsis thaliana plants were obtained with false positive results.Conclusion: In this study,a jasmonic-induced gene TwRIP1 containing the RIP domain was cloned from triticale.It contained five amino acid residues that constituted the active site of rRNA N-glycosidase in the RIP conserved domain.It was predicted that there was no signal peptide and transmembrane domain,and TwRIP1 was located in the cytoplasm.The expression of TwRIP1 was up-regulated under drought stress and MeJA treatment,and it was found that TwRIP1 was also induced by salt stress,low temperature and abscisic acid.It was found that TwRIP1 gene of triticale could regulate the content of proline,change the relative water content of leaves,increase the activity of antioxidant enzyme SOD,improve the photosynthetic capacity and enhance the drought resistanceby using VIGS technology mediated by BSMV virus.At the same time,transgenic Arabidopsis thaliana and wheat plants were obtained,which laid a foundation for further analysis of the function of this gene.