Vector Construction Of OsWHY1 Gene In Rice And Study On Its Correlation With Stay-green Characters

Whirly protein is a ss DNA binding protein found in the nucleus and plastids.As a transcription factor,it can in response to a variety of environmental stimuli by regulating gene expression.Rice is the main food for most of the world’s population,and increasing its yield is of great significance.Under the circumstances that the planting area is limited,increasing the yield of per rice is one of the solutions to the problem.Stay green refers to the traits of plants that remain green and delay senescence in the later stages of growth.As a good trait of plants,it can delay senescence,enable plants to perform more photosynthesis,accumulate more dry matter,increase yield.Therefore,it is of great theoretical significance and practical value to explore the function of rice Whirly protein in stay green trait.In this study,the rice Whirly protein gene(OsWHY1)was cloned from the Oryza sativa indica Jin23 B cDNA.The gene was analyzed using bioinformatics methods.OsWHY1 gene over-expression and interference vectors were constructed,and the expression vectors were transformed into premature senescence rice Jin23 B and stay green rice R287.The chlorophyll content of the transformed plants was determined at three stages: tillering stage,heading stage and filling stage.Drought stress-induced senescence experiments were performed on Jin 23 B T1 generation transformed plant seedlings.The specific results are as follows:(1)Based on the predicted sequence of OsWHY1 gene(LOCUS:XM＿015785850)on NCBI,primers were designed.The RNA of Jin23 B was extracted,and cDNA was obtained by reverse transcription.The full-length cDNA of OsWHY1 gene was 1250 bp by PCR.Bioinformatics analysis of this gene revealed that its open reading frame is 825 bp,encoding274 amino acids.Analysis of protein sequence alignment showed that the similarities of Whirly1 protein sequence with Zea mays,Setaria italica,Panicum miliaceum,Sorghum bicolor and Hordeum vulgare were 79.8%,83.1,%,82.7%,78.7% and 75.3%.(2)The p U1300-OsWHY1 overexpression vector and PTCK303-OsWHY1 RNA interference vector was successfully constructed by restriction enzyme digestion and ligation methods.The expression vector was transformed into indica rice 23 B and R287 through Agrobacterium-mediated genetic transformation.After positive identification of hygromycin primer PCR and identification of OsWHY1 gene expression,a total of 6 strains of Jin 23 B and R287 overexpression,11 strains of Jin 23 B interfered expression,and 13 strains of R287 interfered expression were obtained.(3)The chlorophyll content tester SPAD-502 was used to determine the chlorophyll content in the leaves of each transformed plant and wild-type plant in the three stages of tillering,heading and filling.The chlorophyll of leaves of each transformed plant and wild-type plant was extracted with a mixed solution of acetone and absolute ethanol in the same volume during the filling stage.The absorbance of the extract at 645 nm and 663 nm was measured spectrophotometer and the chlorophyll content was calculated.The results showed that OsWHY1 gene interfered expressing plants had higher chlorophyll content,while OsWHY1 gene over-expressing plants significantly reduced chlorophyll content.(4)Drought-induced senescence was caused by water cut treatment on the seedlings of the Jin23 B T1 generation transformed plants which were transplanted to the greenhouse.Compared with wild-type plants,OsWHY1 gene over-expressing plants showed earlier senile phenotype,while OsWHY1 gene interfered expressing plants delayed senescence.The qRT-PCR was used to analyze the expression levels of senescence-related genes OsGS1,drought-related genes OsDHN1,OsLEA3,drought-related transcription factors Osb ZIP23,OsGRAS23,and OsNAC10 in water cut treated leaves at 0d,19 d and 25 d.OsWHY1 gene over-expressing plants had a large change in gene expression,while OsWHY1 gene interfered expression plants had a lower change.The above experimental results will provide a theoretical basis and transformation materials for further studying the molecular mechanism of OsWHY1 gene in the stay green trait of rice.