Cloning The Key Gene AcICS1 Of SA Biosynthetic And Analysis The Bacterial Cancer Resistance In Kiwifruit

Salicylic acid(SA)is an important plant hormone,which is closely related to plant disease resistance.Isochorismate synthase(ICS)is an important gene of plant salicylic acid synthesis pathway,including ICS1 and ICS2.ICS1 plays a major role in the conversion of branched acids to heterobranched acids.At present,there is no reports about the function of AcICS1 gene in kiwifruit.By analyzing the transcriptome data of kiwifruit,the expression of ICS1 gene was obviously up-regulated in the leaves induced by Pseudomonas syringae pv.Actinidiae(Psa).Meanwile,the content of endo-salicylic acid(endo-SA)in the leaves of ’Hongyang’(susceptible)and ’Jinkui’(resistant)were determined by Liquid Chormatograph Mass Spectrometer(LC-MS),and the results showed that Psa-innoculation were enhanced the production of endo-SA.In this study,kiwifruit AcICS1 gene was cloned and analyzed by using the whole genome sequence of kiwifruit,and the functions of AcICS1 gene were verified by a series of methods including vector construction,instantaneous expression,in situ hybridization and transgene.It laid a foundation for further analysis of AcICS1 gene of kiwifruit in the synthesis of salicylic acid and the regulation of disease resistance.Main results are presented below:1.Determination of salicylic acid in kiwifruit leaves:LC-MS results showed that the content of SA increased when kiwifruit was infected by Psa.That is,up-regulated expression of AcICS1 gene leads to rapid accumulation of salicylic acid.2.AcICS1 gene expression in kiwifruit:Real-time quantatitive PCR was used to quantitatively analyze AcICS1 gene in kiwifruit after Psa-innoculation.The results showed that AcICS1 gene expression was significantly up-regulated after 3 hours of Psa infection.Meanwhile,the expression of AcICS1 gene in Hongyang(susceptible)was down-regulated.3.AcICS1 gene expression in kiwifruit tissues:the expression of AcICS1 gene in different tissues of ’Hongyang’ kiwifruit was detected by in situ hybridization(FISH).AcICS1 expression was found in roots,stems and leaves.AcICS1 gene expression in roots,stems and leaves of kiwifruit inoculated with kiwifruit ulcer fluid was higher than that of uninoculated.This indicated that AcICS1 gene could be upregulated by the pathogen of Psa.4.Cloning and biological analysis of AcICS1 gene of kiwifruit:the full length cDNA sequence and promoter sequence of AcICS1 gene of ’Hongyang’ and Jinkui’ were cloned respectively.The full length of the coding region gene was 1710 bp,encoding 569 amino acids.AcICS1 gene has Chorismate_bind domain,including 256 amino acids(287-543).The overexpression vector and P1300 recombinant vector were constructed respectively.The promoter sequences contain typical core elements of eukaryotic gene promoters TATA box and CAAT box,and a variety of cis-elements related to light regulation,stress and plant hormones.The promoter of ’jinkui’ AcICS1 gene has TCA-element which responds to salicylic acid5.Determination of salicylic acid in tobacco leaves:The content of SA in the tobacco leaves injected with P1300 recombinant carrier solution of AcICS1 gene was 1 times higher than that in the leaves injected with P1300 empty carrier solution.In contrast,JA decreased by 0.25 times.Overexpression of AcICS1 gene led to up-regulated expression of SA and down-regulated expression of JA.6.Genetic transformation of plants:set up a stable regeneration system of kiwifruit,and carry out PCR detection on the plant extracted DNA on the screening medium,and get 3 plants of positive plants that are super expressed by jinkui AcICS1 gene.