Cloning And Functional Analysis Of GhSKIP35 Gene Responsiblefor Resistance To Verticillium Wilt In Gossypium Hirsutum L.

As an important economic crop,cotton plays an important role in our national economy. Verticillium wilt has become one of the most devastating disease leading to serious loss of cotton production.The usage of wilt-tolerant/resistant cultivars becomes the primary method to manage this disease.So,through research on the molecular mechanism during the cotton defense response to V.dahliae and identification of defense related genes has become the main direction of future cotton breeding.Researchs reveal SCF as an important ubiquitin-protein ligating enzyme E3 plays a role on defense response by mediating the resistance of R gene,Jasmonic acid and ethylene response.This study bases on a suppression subtractive hybridization based cDNA library constructed from V.dahliae inoculated KV1.Detailed functional identification of GhSKIP35 gene from SCF is studied. The main results of this study are as follow:A new gene named GhSKIP35 is isolated from Zhongzhimian KV3 by using gene electronic cloning,3’race and 5’race.The open reading frame(ORF) is 1863 nucleotides(nts) long, which encodes a protein of 620 amino acid. With the amino acids sequence alignment of Arabidopsis thaliana, Cicer arietinum, Citrus sinensis, Cucumis melo, Cucumis sativus, Malus domestica, Prunus mume, Solanum lycopersicum, Solanum tuberosum, Vitis vinifera; GhSKIP35 has 76.63% overall homology and the completely same conserved domain. It is most closely related to Citrus sinensis SKIP35 protein by phylogenetic analysis.The putative protein contains a conserved ANK domain as long as 100 amino acid.Real-time quantitative reverse transcription polymerase chain reaction shows that GhSKIP35 gene is activated quickly after inoculation with Verticillium dahliae. It reachs the peak point at 12 h post-inoculation(hpi), and then its expression is gradually decreased within 24-48 h. These results confirm that GhSKIP35 is up-regulated gene.It is assumed that GhSKIP35 might play an important role in the defense reaction against Verticillium wilt.Virus induced gene silencing is used to silence endogenous genes in resistant upland cotton cultivar Zhongzhimian KV1 via targeting the fragment of GhSKIP35. V991 is used to inoculate silence cotton plants in order to identify disease resistance. The results show that the disease indices of wild type Zhongzhimian KV1 and VIGS with vector control plants are 9.38 and 11.54 respectively. The disease indices of silencing GhSKIP35 plants is 50.75. These results confirm that GhSKIP35 gene was related to Verticillium resistance in cotton.We construct a plant over-expression vector of GhSKIP35 Gene, and transfer it into Arabidopsis thaliana by Floral Dip method. Over expression of the GhSKIP35 gene endows transgenic Arabidopsis with higher resistance to V. dahliae.These results furtherly confirm that GhSKIP35 gene was related to Verticillium resistance.Our results demonstrate that the GhSKIP35 gene is responsible for resistance to V. dahliae in island cotton and can be used for breeding cotton varieties that are resistant to Verticillium wilt.