| Phosphorus is one of the core life elements. Clarifying the function of phosphate regulated gene is helpful to identify phosphate high efficient-used genes and understand gene expression-nets of phosphorus metabolism. At5g55690 is a MADs-box protein family transfaction factor gene, ecoding AGAMOUS-LIKE 47(AGL47) protein. MADs-box protein family gene is key-regulation factors both in planta vegetative growth and reproductive growth. Early studies have shown that the expression of at5g55690 gene is specific regulated by phosphate stress.So it’s lilely to play an important role in phosphate regulatory pathway. To further identify the function of the at5g55690 gene. In this study, we are doing the physiological measurements of over-expression mutants that cultured in hydroponic sytems. Eestablished the alcohol induction over-expression test systems, and completed the AGL47 protein Prokaryotic expression, and get the basis to clarify the function of this gene. The main results are as follows:1. Physiological measurement for over-expression mutants of at5g55690 gene of A. thaliana(1) Detection of over-expression mutant. Former study created over-expression seeds sown on 1/2 MS medium which contain hygromycin antibiotic to select hygromycin resistant mutants. Specific PCR identified that two mutants two is positive mutant, Semi-quantitative detected that at5g55690 gene expressed highly in vivo in mutants.(2) Over-expression at5g55690 gene promoted the vegetative growth and delayed plants bloting. When A.thaliana growed at 10μM phosphate hydroponic system, compared to wild-type, mutant bloting and pod are all delayed for one day. When A. thaliana growth at 500μM phosphate hydroponic system, wild-type bloting at 14th day, pod at 16th day, bloting ratio at 16th day is 100%; Mutant bloting at 14th day, but until 16th day, there was not pod,and bloting ratio at 16th day is only 90%.(3) Over-expression at5g55690 gene enhanced seedings growth at low-Pi. When A.thaliana growed at 10μM phosphate hydroponic system, compared to control wild-type A.thaliana, mutant fresh wight increased 123.70%, drought weight increased 91.38%, root length increased 19.50%,root drought increased 52.46%. When A.thaliana growth at 500μM phosphate hydroponic system, compared to control wild-type A.thaliana, mutant fresh wight increased 93.1%, drought weight increased 77.78%, root length increased 27.99%, root drought increased144.57%.(4) Over-expression at5g55690 gene increased the phosphate uptake and affected the distribution of phosphorus in plant. When A. thaliana growth at 10μM phosphate hydroponic system, compared to control wild-type A.thaliana, phosphate uptake of mutants increased 96.33%. When growed at 500μM phosphate hydroponic system, compared to control wild-type A.thaliana, phosphate uptake of mutant increased 53.88%. Over-expression at5g55690 gene made phosphous content in mutant root and shoot change smaller than wild-type. When growed at 10μM phosphate hydroponic system, mutant shoot contains 4.55 times phosphorus than root, wild-type shoot contains 5.90 times phosphorus than root. When A.thaliana growth at 500μM phosphate hydroponic system, mutant shoot contains 4.01 times phosphorus than root, wild-type shoot contains 1.74 times phosphorus than root.(5) Over-expression at5g55690 gene reduced the physiological sensitivity to low phosphate stress. When A.thaliana growth at 0μM and 10μM phosphate hydroponic system, root/shoot ratio of mutant smaller than wild-type A.thaliana. Over-expression at5g55690 gene promotes root growth, so when growed at 500μM phosphate hydroponic system, root/shoot ration of mutant higher than wild-type A.thaliana.2. Development of alcohol induced at5g55690 over-expression experimental system. Zheng Wenming. et al (2007) had cloned at5g55690 full-length ORF to alclhol induced over-expression vector pSRN, constructed recombinant vector PSRN-at5g55690, transfected into Arabidopsis thaliana by Agrobacterium and create alcohol induced over-expression at5g55690 gene mutant.In this study, we used different Alcohol concentration and different times to induce mutant express at5g55690 gene in vivo and using Semi-quantitative RT-PCR detected at5g55690 gene expression. The results showed that 0.25% alcohol induction mutant is the most appropriate, and inducrion for about two hours could get best effect, then the Seedling could be harvest and used as the material for next step chip-sequence experimental.This is good basis for future study it’s downstream target genes and interacting proteins.3. AGL47 protein prokaryotic expression. at5g55690 gene full-length ORF was cloned into the expression vector to construct recombinant vector pet-28a-at5g55690. The recombinant vector then was transformed into E. coli DH5αand select of positive strains and extract plasmid, carry out double digestion and sequencing identify. Recombinant vector transformed into E. coli BL21 (DE3).Under IPTG induction, Achieved AGL47 protein expression in prokaryotic cell. Fusion protein exists in the form of inclusion body. Through the inclusion body purification, denaturation and renaturation,initially got the soluble fusion protein. |
PDF Full Text Request