The Role Of Orphan Gene OsCPY In Chilling Tolerance Of Rice

Low temperatures severely affect the growth and development of rice and reduce rice yield.An effective way to increase rice yield is to understand the molecular mechanisms of rice responses to low temperature stress,to study the functions of rice genes associated with resistance to cold stress,and to use molecular biology methods to improve rice genetic characteristics.We used Gateway technology to construct a cDNA library of drought-and salt-stressed rice roots and discovered a new orphan gene in it.We named it OsCPY.In this study,we carried out the bioinformatics analysis of OsCPY gene coding sequence and promoter,analyzed the expression of OsCPY under abiotic stress by qRT-PCR,we constructed the interference vector of OsCPY gene,and transferred it to rice,and obtained OsCPY down-regulated transgenic rice.Phenotypes of transgenic rice with OsCPY over-expression or down-regulated were observed and analyzed under low temperature stress.The main results were as follows:1.Bioinformatics analysis of OsCPY geneBioinformatics analysis of the OsCPY gene using the rice genome database of the online phytozome website revealed that OsCPY is located on chromosome 9 and contains only two exons and one intron.According to the PlantCSRE online tool,the cis-acting element of OsCPY promoter was analyzed.The results showed that the promoter of the gene has four abiotic stress-related components:one heat stress-related element HSE,one GA stress-related element TATC-Box,two ABA stress-related elements ABRE,motif IIb.In addition,there is also a cis-acting element Skn-1-motif associated with endosperm expression in the OsCPY promoter.2.Expression of OsCPY gene in different tissues of riceTo understand the expression characteristics of the OsCPY gene,we used qRT-PCR to analyze the expression of OsCPY in different tissues of rice under normal growth conditions.The results showed that under normal growth conditions,OsCPY gene was expressed in roots,stems,leaves and young panicles of rice,and the expression level was highest in leaves.The expression levels were 3.38 times as much as the root and 4.38 times as much as the stem.In the first ten days of flowering,the expression of OsCPY gene showed a downward trend with the increase of flowering time.On the tenth day,the expression level of OsCPY gene was reduced to21%of the third day.3.Expression analysis of OsCPY gene under stressZhonghua 11 was treated with low temperature,high salt,and drought stress,and the expression of OsCPY in each material was analyzed.The results showed that under low temperature stress,the expression of OsCPY gene in roots,stems and leaves increased by 5.80,4.29 and 5.07 times,respectively.Under high salt stress,the expression of OsCPY gene was increased by 3.89 times in stems and 2.44 times in roots.Under drought stress,the expression of OsCPY gene was increased by 4.60times in stems and 2.47 times in roots.4.Construction of OsCPY interference vector and rice transformationIn order to study the effect of OsCPY expression down-regulation on rice,we constructed an interfering vector for the OsCPY gene using the PYL-RNAi plasmid,named CPY-RNAi.The CPY-OE and CPY-FC OsCPY gene expression vectors were constructed in the laboratory and were transformed into Zhonghua 11 using Agrobacterium-mediated method to obtain the CPY-RNAi transgenic rice.There were30 plants,27 CPY-OE transgenic rice plants,and 35 CPY-FC transgenic rice plants.The expression levels of OsCPY transgenic plants were analyzed using qRT-PCR,and three lines of T₂ generation homozygous plants were identified.Compared with the wild type,OsCPY expression levels in the down-regulated expression plants?RNAi-14,RNAi-74,RNAi-36?were 16%,30%,and 35%of Zhonghua 11respectively.In OsCPY-overexpressing plants?OE-24,OE-27,OE-14?,the expression levels of this gene were 3.75,3.83,and 2.41 times as much as Zhonghua 11,respectively;In functional complement plants?FC-35,FC-13,FC-15?,the expression levels of this gene increased by 3.82,2.23,and 2.10 fold,respectively.5.Observation of growth and development phenotypes of OsCPY transgenic plants under low temperature stressTo understand the role of the OsCPY gene in low-temperature tolerance of rice,growth of homozygous OsCPY transgenic lines and the OsCPY mutant?cpy?were observed.The results showed that after 5 days of low temperature stress at 8°C,the wild-type control leaves appeared curling,the leaves were dull,the cpy and CPY-RNAi leaves were stretched and the color was green,and the growth was better.The leaves of CPY-FC plants showed slight crimping.CPY-OE plants had severe leaf curling.After normal temperature was restored,CPY-RNAi plants could continue to grow.CPY-FC plants had new leaves,but the growth was weak,and CPY-OE plants withered or even died.6.Detection of germination rate of rice seeds mediated by ABA and GAUnder 15°C low temperature stress,the seeds were treated with different concentrations of ABA.The results showed that there was no significant difference in the germination rate of mutant cpy and wild-type seeds.After treatment with 1?M,10?M,and 100?M GA,the germination rate of mutant cpy seeds was increased by18.62%,9.89%,and 9.05%,respectively,compared with wild-type rice.And there was a significant difference in the germination rate between the two,indicating that the OsCPY gene may inhibit GA in the role of seed germination.