Cloning And Functional Analysis GhbHLH130 And GhZFP130 In Cotton (Gossypium Hirsutum L.)

Abiotic stress such as high salinity, drought and low temperature are major obstacles, which affecting crop growth and productivity. During the long evolution, the plant has formed a set of mechanisms to adapt or fend off stress injuries. As a result, a number of researchers tried to study the function of crucial genes and their regulation during plants responsiveness to stress. It is important meaning to achieve enhanced stress tolerance. So far, the separation and identification of genes related to stress that up to hundreds in plants, that including functional genes and regulatory genes. Among them, the bHLH transcription factor family and zinc finger protein family are widely response to stress in plant.1. We cloned two genes related to abiotic stress such as GhZFP130 and GhbHLH130 in upland cotton for the first times. Sequence and homology comparison revealed that GhbHLH130 gene encodes a bHLH family transcription factor, the amino acid sequence showed a very typical HLH domain. Cluster analysis showed that bHLH transcription factors in the cocoa, soybean and rice have close genetic relationship. GhZFP130 encodes a Ziml7 type zinc finger protein, what belongs to a poorly studied zinc finger protein family. Amino acid sequence showed GhZFP130 is a typical Zim17 type zinc finger protein which has a very typical zf-DNL domain, multiple sequence alignment suggested Zim17 type zinc finger proteins in cocoa, rice, yeast and other species have conserved domains. the protein as assisting protein transport other proteins into the mitochondrial matrix, there are few studies of this type zinc finger protein adapt to abiotic stress. Analysis showed the presence of two transmembrane domains (TM) in the 64-82 amino acids and the 142-160 amino acids. Taken together, GhbHLH130 and GhZFP130 may play important roles in cotton responsiveness to abiotic stresses.2. GhZFP130-GFP fusion protein was constructed and transiently expressed in onion epidermal cells, the green fluorescence was detected in mitochondria of onion epidermal cells. GhbHLH30-GFP fusion protein was transiently expressed in onion epidermal cells, the green fluorescence was detected in the nucleus of onion epidermal cells.3. Q-PCR analysis showed that GhbHLH130 and GhZFP130 by high salt,drought,cold and ABA -induced, but have different induction modes. Using PEG6000 treated cotton roots, GhbHLH130 gene expression level appear the supreme state after 3 h PEG6000 treatment, after 6 h PEG6000 treatment expressing quantity significantly reduced. After NaCl and exogenous ABA treatment, gene expression pattern as same as PEG6000 treatment. There were increased first, then decreased, and then expressed in 6 h appeared a peak. After 6 h cold treatment, GhbHLH130 gene up to highest expression level. GhZFP130 gene express patterns response to stress are different from GhbHLH130. Using NaCl solution to irrigate cotton roots, GhZFP130 gene expression quantity appear a peak at 6 h, and then is on the decline. After 6 h cold treatment, the gene expression quantity reach the highest level. After 3 h PEG6000 treatment, GhZFP130 express quantity is highest. GhZFP130 gene strongly expressed after 6 h exogenous hormone ABA treatment. GhZFP130 and GhbHLH130 genes might be used to engineer the biotech crops with tolerance to stress.4. We construct PBI121-GhbHLH130 overexpression vectors, transfer the GhbHLH130 gene into tobacco to verify the function of the gene, and extracted the DNA and RNA of the 8 transgenic tobaccos, from which we knewn that we have succeed in getting 8 transgenic tobacco. We treated the transgenic ones and wild type tobaccos with drought stress for 15 days finding the ability of drought resistance is higher than the WT. And construct PBI121-GhZFP130 overexpression vectors, transfer the GhZFP130 gene into Arabidopsis thaliana to verify the function of the gene, we have succeed in getting 6 transgenic Arabidopsis thaliana. To compare the wild type and GhZFP130 transgenic arabidopsis tolerance to drought, test the rate of water loss of vitro leaf under natural conditions. We found that transgenic GhZFP130 Arabidopsis filtration rate is lower than the wild type. The results showed that the transgenic plants have higher resistance than the wild type.