Functional Analysis Of Three Zinc-finger Proteins (ZFPs) Under Different Stress Treatments By Virus-induced Gene Silencing (VIGS) In Cotton(Gossypium Hirsutum L.)

Cotton is one of the important strategic materials which concerns the national economy and people’s livelihood,and it has an important position in the pattern of agricultural economy.Cotton production of China is ranked among the top of the world,and China is also the biggest cotton consumer.With the deteriorating environment,abiotic stresses such as drought,high salinity,extreme temperatures,restrict the improvement of yield and quality of cotton seriously;The global arable land is decreased,and recent years,our country adjusts the planting industry structure,the cotton planting area is reducing yearly.Cotton is a crop with strong resistance to abiotic stress,thus to further improve the ability of cotton resistance to abiotic stress,is an important way to improve the land utilization and ensure the cotton production.Zinc finger protein is a kind of transcription factor which can combine with zinc ions and folds into finger structure domain,it is widely distributed in plants,animals,and microorganisms.This kind of protein regulates the expression of downstream genes by combining with DNA,RNA or interacting with other proteins,its role throughout every process of life activity.Zinc finger protein of plants plays an important role in its growth and stress resistance.Virus induced gene silencing(VIGS)is a technology that infects plants by recombinant virus vector carrying a purpose gene sequence,after infecting,the purpose gene will silence and then the plants’ phenotype changes,then we can analyse gene function through the phenotypic variation.This technology can be used to analyse plant gene function fast and efficiently.We got three zinc finger protein genes,GhZFP2,GhRCHY1 and GhBBX1 by homology cloning earlier,their expression analysis under different abiotic stresses were detected by qRT-PCR,transgenic results showed that these three genes can improve the resistance to salt and drought resistance of tobacco.Based on previous research results.in this study we built VIGS carriers and silenced the genes respectively in cotton,then the cotton plants were treated with salt stress and drought stress after infecting,the genes’function were analysed by measuring the related physiological indexes;We also built the rapid functional verification system of genes associated with cotton abiotic stress using VIGS technology.The main results as follows:1、The Jinmian19 cotton seedlings were screened with different concentrations of NaCI and PEG6000 and the optimization concentration of NaCl treatment was 200mM,the optimization concentration of PEG6000 treatment was 15%(w/v).We got the pTRV2-GhZFP2,pTRV2-GhRCHY1,pTRV2-GhBBX1 recombinant vectors by building the purpose gene segment to pTRV2 carrier,then transferred them to cotton by infecting.The condition of the purpose gene silencing were detected through qRT-PCR and RT-PCR,and silent plants with purpose gene expression obviously reduced were obtained.2.The silent plants were treated with salt stress,five indexs related to salt tolerance,including net photosynthetic rate,Pro content,SOD activity,MDA content and K+/Na~+,were measured respectively.The results of the correlated indexs showed:under salt stress,the leaf net photosynthetic rate of gene silencing plants were significantly lower than the Mock group,and the pTRV2-GhZFP2,pTRV2-GhRCHY1 reached the high significant level;The change ratio of Pro content of all silent plants was lower than the Mock group,and pTRV2-GhBBX1 group reached the significant level;The change ratio of SOD activity of all silent plants was highly significant lower than the Mock group;The change ratio of MDA content of all silent was higher than the Mock group,but did not reach significant level;The K+/Na~+ of aerial part of all silent plants was significantly lower than that in Mock group,and pTRV2-GhBBX1 group reached the highly significant difference,the K+/Na~+of underground part of pTRV2-GhRCHY1,pTRV2-GhBBX1 group was significantly lower than the Mock group.The results showed that under salt stress,net photosynthetic rate of gene silencing plants is lower than the Mock group;accumulation rate of osmotic regulation substances is lower than the Mock group;Ros changes of enzyme activity is lower than the Mock group,at the same time accumulation rate of membrane oxidation product is higher than the Mock group;aerial part and underground part absorb and accumulate more Na~+,thus destroy the nutritional balance.3.The silent plants were treated with drought stress,six indexs related to drought tolerance,including net photosynthetic rate,leaf water loss rate,stomatal aperture,Pro content,SOD activity,MDA content,were measured respectively.Drought-resistant related index test results show that:under drought stress,the leaf net photosynthetic rate of gene silencing plants were lower than the Mock group,and pTRV2-GhZFP2,pTRV2-GhRCHY1 reached the significant level;water loss from detached leaves of all the silent plants was higher than the Mock group after drought stress;stomatal aperture of all silent plants was highly significant smaller than the Mock group after treating with drought stress for 10 days and 20 days;The change ratio of Pro content of all silent plants was lower than the Mock group,and pTRV2-GhZFP2,pTRV2-GhRCHY1 group reached the significant level;The change ratio of SOD activity of all silent plants was significantly lower than the Mock group;The change ratio of MDA content of all silent was higher than the Mock group,and pTRV2-GhBBX1 group reached significant difference.The results show that under drought stress,net photosynthetic rate of gene silencing plants is lower than the Mock group and the photosynthesis is influenced;water loss rate is higher than the Mock group and stomatal aperture is smaller than the Mock group;accumulation rate of Osmotic regulation substances is less than the Mock group,thus reduce the osmotic regulation ability;Ros changes of enzyme activity is lower than the Mock group,at the same time the accumulation rate of plasma membrane oxidation product is higher than the Mock group,thus the integrity and stability of the plasma membrane is undermined.Our Research shows that the three genes possess important role in response to salt stress and drought stress,at present the cotton genetic transformation work of the three genes is under way.