Molecular Mechanism Of Rice RIP2 In Leaf Angle Regulation

The leaf angle is an important part of the plant architecture of rice,which is related to the tolerance to the stress,photosynthetic efficiency and the degree of planting density,so as to ultimately affect the yield of rice.Therefore,it is an important problem for molecular breeding to find out the molecular mechanism of leaf angle formation and then drive the precise change of leaf angle through biotechnology.Our team has cloned the rice leaf morphogenetic gene rolled and erect leaf 1(REL1).The expression of REL1 can lead to the phenotypic variation such as leaf curl and leaf angle increase,and the significant increase of leaf paraxial vesicular cells,and it can participate in the regulation of leaf angle through the Os BZR1 gene in the synergistic BR pathway.On this basis,REL1 interactive protein RIP2(REL1interactive protein)was used in this study.As the research object,the interaction of RIP2 and REL1 in vivo and in vitro was confirmed by yeast two hybrid and bimolecular fluorescence complementation;the molecular mechanism of RIP2 regulating the formation of leaf angle was preliminarily determined by bioinformatics,expression mode,subcellular localization,knockout and overexpression;and the molecular mechanism of rice gene RIP2 regulating the formation of leaf angle was further revealed by rel1rip2 double mutant and exogenous BR treatment and the whole genome expression profile.The main results are as follows:1)p GADT7-RIP2 and p Gbk T7-REL1 co-transformed yeast grew in QDO(quadruped dropout supplies)plate,indicating that REL1 interacted with RIP2 protein in vitro;p SPYCE-RIP2 and p SPYNE-REL1 co-transformed rice protoplast,and the yellow fluorescent protein signal fused with the spontaneous fluorescence signal of chloroplast,indicating that REL1 interacted with RIP2 protein in vivo and in chloroplast.2)Bioinformatics analysis showed that RIP2 was 2212 bp in length,composed of four exons and three introns,741 bp in CDS encoding 246 aa,and RIP2 was a stable hydrophilic protein with a conserved domain RING＿Ubox。3)The results of q RT-PCR showed that the expression of RIP2 was the highest in the underground part of seedling stage,and higher in the leaf sheath,leaf pillow and leaf tongue of booting stage;subcellular localization showed that RIP2-GFP fusion protein signal fused with Golgi maker,indicating that RIP2 protein was located in Golgi body.4)Compared with wild-type ZH11,the leaf angle of RIP2 knockout material increased significantly,indicating that RIP2 gene negatively regulated the leaf angle;the leaf angle of RIP2 OE was close to 0,but there was significant difference between RIP2 OE and wild-type ZH11.5)Compared with the mutant rel1,the leaf angle of the two mutants rel1rip2 increased to a significant level,which indicated that the effect of the two mutants could be accumulated,and REL1 and RIP2 might be one of the antagonistic factors to control the leaf angle.6)The results of exogenous BR showed that the leaf angle of wild-type ZH11 and RIP2 OE increased significantly with the increase of BR concentration,and showed a dose-dependent effect,while the leaf angle of rel1,rip2 and rel1rip2 had no significant change before and after the application of BR,indicating that they were not sensitive to the BR.In addition,q RT-PCR was used to detect the expression changes of REL1 and RIP2 in ZH11 treated with exogenous hormones.It was found that the expression of REL1 increased rapidly after BR,while the expression of RIP2 decreased rapidly,which indicated that exogenous BR could induce the expression of REL1 in rice and inhibit the expression of RIP2,which was in m RNA;At the same time,REL1 and RIP2 were proved to be antagonistic factors to regulate the angle between leaves.7)A total of c DNA libraries were constructed and analyzed 77.41 G sequence data:there are 41 differentially expressed genes in ZH11 and rip2,zh11 and rel1,rip2 and rel1rip2,and rel1 and rel1rip2.In the comparison of ZH11 and rip2,30 positive regulatory genes and 22 negative regulatory genes were screened and identified.At the same time,some genes related to Brassinolide(BR)and auxin(IAA)synthesis and conduction were identified The results of q RT PCR showed that the expression of RIP2 was basically consistent with the whole genome expression profile data,indicating that the leaf angle regulated by RIP2 was related to the synthesis and signal transduction of BR and IAA.This study provides the experimental basis for the analysis of the molecular mechanism of RIP2 involved in the regulation of leaf angle,and provides the basis for the exploration of the genetic effect on the development of leaf morphology.