Study On The Regulation Of ZmSPRP1 On Starch Synthesis In Endosperm And Photosynthesis In Leaf Of Maize

Maize is one of the most widely cultivated crops in the world.Maize starch synthesis is a physiological activity mainly in endosperm,which directly affects maize yield and is the key to the formation of "sink";maize photosynthesis is the process of transforming light energy into chemical energy mainly in leaves,which is the basis for the formation of "source".It has been shown that starch synthesis and photosynthesis related gene expression in maize are regulated by some transcription factors.On the other hand,a large number of RNA binding proteins have been found to play a role at the transcription level just like transcription factors.At present,there is no research on the transcription factors that can promote both endosperm starch synthesis and leaf photosynthesis,and there is no clear report that RNA binding proteins in plants participate in transcription regulation.In this study,we found that a RNA binding protein Zm SPRP1 not only promoted the activity of key enzyme promoter of starch synthesis in endosperm,but also promoted the expression of a large number of photosynthesis related genes in leaves.This study also verified the binding characteristics of Zm SPRP1 to RNA.This study is helpful to further improve the regulation network of starch synthesis and photosynthesis transcription,and provide a theoretical basis for the increase of maize yield by "expanding source and sink".The main results are as follows:1.We obtained 45 genes encoding KH domain protein and 299 genes encoding RRM domain protein in maize genome by bioinformatics analysis.By analyzing the RNA-seq data of maize tissues,we found that 11 genes encoding KH protein and 14 genes encoding RRM protein had the highest expression in endosperm,and some of them were also highly expressed in maize leaves.We selected five genes from each of the two types of RNA binding proteins as candidates for further study.2.In order to further screen the RNA binding proteins which can promote the activity of starch synthesis related gene promoters,we cloned 10 coding regions of RNA binding protein genes into PBI221 transient over expression vector.At the same time,the report vector(promoter of corn starch synthesis related gene driving Luc expression)constructed by our lab was used.We bombarded the young endosperm of Maize with gene gun,and screened a protein containing KH domain from 10 candidate RNA binding proteins,which can promote the activity of promoter of starch synthesis related genes extensively,and named the protein Zm SPRP1.The high expression of Zm SPRP1 gene in endosperm was found by RNA in situ hybridization.Semi-quantitative analysis showed that the expression of Zm SPRP1 gene was the highest in grains and also in leaves.3.In order to further search for the DNA segments in the promoter of starch synthesis related genes that respond to the regulation of Zm SPRP1,we constructed the promoter deletion vectors of Zm GBSSI and Zm BT1 genes,which drive Luc expression by the promoters of the missing segments.A 218 bp region in the Zm BT1 promoter were found to respond to the regulation of Zm SPRP1.Then,we further verified the binding characteristics of Zm SPRP1 to these two regions by yeast one hybrid experiment.The results show that Zm SPRP1 can bind to the 218 bp segment of Zm BT1 promoter.4.In order to verify whether Zm SPRP1 can be located in the nucleus,we constructed a vector expressing Zm SPRP1-e GFP fusion protein,and bombarded the inner epidermal cells of onion with gene gun.The results showed that Zm SPRP1 was distributed in the cell nucleus,cytoplasm and membrane of onion.Then,we transformed the vector expressing Zm SPRP1-e GFP fusion protein into leaf protoplast.The results showed that Zm SPRP1 could be located not only in nucleus,cytoplasm and cell membrane,but also in chloroplast.In order to study whether Zm SPRP1 can regulate gene expression in leaf cells,we transformed the vector over expressing Zm SPRP1 into leaf protoplast and sequenced the transcriptome.The results showed that Zm SPRP1 could promote the expression of a large number of genes related to photosynthesis,and many of the up regulated genes belonged to chloroplast genome.The localization of Zm SPRP1 in chloroplast is consistent with its function of promoting gene expression in chloroplast.Subsequently,we constructed a report vector which Luc expression were driven by some up-regulated gene promoters,and transformed the leaf protoplasts together with the vector over expressing Zm SPRP1.The results showed that Zm SPRP1 could enhance the promoter activity of these photosynthesis related genes.5.In order to search for the interacting protein of Zm SPRP1,we obtained some proteins related to transcription and post transcriptional regulation through screening yeast two hybrid library.Among the three interacting proteins,one is RNA binding protein with KH domain;the other encodes a 5 ’-3’ exonuclease;in addition,Zm SPRP1 can interact with itself and form homologous dimer.We found that the interacting RNA binding protein can also promote the activity of Zm BT1 promoter.6.In order to verify the binding ability of Zm SPRP1 to RNA,we carried out yeast three hybrid experiment.The results showed that Zm SPRP1 was able to bind a section of C-rich RNA and poly(A)RNA.Then,we synthesized 158 bp poly(A)RNA through transcription in vitro,and carried out RNA pulldown experiment on the total protein of maize grain 12 days after pollination,and analyzed the protein obtained from the experimental group and the control group by mass spectrometry.It was found that Zm SPRP1 was one of several proteins with RNA binding domains pulled down by poly(A)RNA.However,the subsequent gene gun experiments did not find that Zm SPRP1 had a regulatory effect on short fragment poly(A).The function of Zm SPRP1 on post-transcription needs further study.7.We obtained zmsprp1 mutant material through Maize GDB maize mutant library.After backcrossing to W22 wild type,the homozygous mutant and wild type plants can be detected in the progeny of selfing.Through the observation of plant morphology at 7-leaf stage,it was found that the mutation of Zm SPRP1 caused the plant growth to be weaker than that of W22 wild type.Further measurement and data analysis of the length and width of the fifth leaf showed that the mutation of Zm SPRP1 led to a significant decrease in the length and width of the fifth leaf.In addition,we found that the yellow degree of the third leaf of the mutant was more serious than that of the wild type.At 12 days after pollination,the expression of Zm Sh2,Zm BT1 and other starch synthesis related genes in zmsprp1 mutant decreased.We created a gene knockout material of Zm SPRP1.It was found that part of the grains in the self crossing ear of the gene knockout plant only formed empty peel,and did not accumulate starch and other contents.Further backcross and self cross and morphological identification are needed for knockout materials.In this study,we investigated the regulatory mechanism of Zm SPRP1 on maize endosperm starch synthesis related genes and photosynthesis related genes at the transcription level through molecular biology technology,which laid the foundation for improving the transcription regulatory network of photosynthesis and starch synthesis.The regulation mechanism of Zm SPRP1 in leaf and endosperm can provide a theoretical basis for the creation of high quality materials and high-yield breeding in future.