Functional Analysis Of Stress Associated Zinc Finger Protien Gene ShSAP1from Sugarcane

Sugarcane (Saccharum officinarum L.) is main crop for sucrose producing. Abiotic stresses especially drought pose serious threats to growth and productivity of sugarcane, it is very important to discover abiotic stress associated genes in sugarcane and uncover their functions, which is basis for cultivation of of new varieties with stress-resistance and high yield by resistant gene transferring. Zinc finger protein is a superfamily that involved in many aspects of plant growth and development in plants. Zinc finger proteins regulate gene expression through interacting with DNA, RNA or protien with their typical zinc finger domains. The Stress Associated Proteins(SAPs) gene family is composed of genes encoding proteins containing A20/AN1zinc-finger domains. A20/AN1zinc-finger domain proteins have been shown to be involved in stress response and confer abiotic stress tolerance in different plants. From sugarcane stalks maturing related cDNA library, we obtained a zinc finger protien gene ShSAPl with A20and AN1zinc finger in its deduced protein. To further understand the life process that ShSAPl would be involved, we analysis its gene structure, expression pattern, promoter and subcellular localization. Then ShSAP1was transmit into tobacco, its effect on tobacco stress tolerance and mature process was analyzed. Function of ShSAP1in sugarcane was studied through inducible expression and silencing of ShSAP1by transgenic approach. The main results are as follows:1. ShSAP1encodes a predicted polypeptide of171amino acids with an A20and AN1zinc-finger domain at the N-and C terminal, respectively, belongs to SAPs(Stress-associated proteins). ShSAP1has the highest amino acid sequence identity with Zeamays ZmAN13and rice OsSAP8. ShSAPl was present as one or two copy in the genome of sugarcane with two introns located in the5’UTR region.1243kb genomic sequence upstream of ShSAP15’ UTR predicted the presence of several cis-acting regulatory elements involved in the stress response such as dehydration responsive element, ABA responsive elements(ABRE), MYB/MYC binding elements.2. ShSAP1expression was observed in the roots, stalks and leaves of sugarcane and much higher in mature part of all organs and highest in maturest internode, significantly low in young leaves and roots. ShSAPl expression was induced by salt, drought, abscisic acid(ABA),ethephon(ET) and gibberellin(GA3) during seedling stage. Subcellular localization of ShSAPl-GFP fusion protien was observed in the nucleus in addition to the cytoplasm and plasma membrane of onion epidermal cells. To confirm the result, we established anther expression vector expressing ShSAPl-GFP-GUS fusion protien and found the fusion protien located in cytoplasm of onion epidermal cells and tobacco protoplast. Soluble ShSAPl-His fusion protein was obtained through prokaryotic expression. After purification, the fusion protein was used to immune rabbits and polyclonal antibody was prepared.3. Transgenic tobacco were obtained using the leaf disc infection method. Using ShSAP1polyclonal antibody, ShSAP1expression was observed in transgenic lines(T4, T16, T21)with low ShSAP1copy. Expression of ShSAP1in transgenic tobacco conferred tolerance to salt and drought at late seedling stage compared to wild-type(WT). The expression of stress-responsive genes, NtAPX, NtCAT, NtTXP, NtPOD, NtERD10A, NtERD1OB, Osmotin and NtERF5were up-regulated in transgenic plants after drought treatment. Transgenic tobacco with ShSAP1have better drought resistance during vegetative stage, maintained better membrane integrity (with lower plasma membrane permeability and MDA content), And showed better water retaining capacity and oxidation resistance (with higher proline content and enzymatic activity of POD and CAT) under drought treatment.4. ShSAP1driven by Prd9A was transmited into sugarcane and two transgenic lines(S5and S14) with higher expression level of ShSAP1were obtained. RNAi expression vector of sugarcane A20/AN1zinc finger protien gene ShSAP1was constructed and transformed into sugarcane, two transgenic Iines(il7and i44) with partial silencing of ShSAP1were obtained. At sugarcane seedling stage, salt and drought tolerance tests were conducted in greenhouse. Transgenic lines with higher ShSAPl expression level exhibited better drought resistance with higher survival rate than control and RNAi lines, and maintained higher net photosynthetic rate and water-holding capacity during drought treatment; Better salt resistance was also observed in S5and S14via chlorophyll determination after salt treatment. According to all above the indexes, RNAi lines showed a little more sensitive to salt and drought stress. By phenotyping in the field, we found transgenic lines with higher ShSAP1level showed higher net photosynthetic rate at elongation stage and have more effective stalks and higher stalk sugar content at mature stage, while all these indexes of silencing lines showed lower than non-transgenic lines. The results indicate ShSAP1can improve drought and salt resistance and may promote the process of growth and sucrose accumulation in sugarcane.