Effects Of ZmKRP6 And ZmKRP8 On The Development Of Endosperm Cells In Maize

Maize(Zea mays L.)is one of the major food crops in the world.It serves as feed,fodder and industrial raw materials.The proportion of endosperm in maize grain is about80%.The development of endosperm will have a direct impact on the grain yield and quality.Studies on cereal crops have shown that protein complex formed by cyclin-dependent kinases(CDKs)and cyclins,plays an important role in the regulation of endosperm development.The inhibitor/interactor of Cdc2 kinases(ICKs),also known as Kip-related Proteins(KRPs),could affect cell division and endosperm development by binding with protein complexes formed by CDKs and cyclins.However,our understanding of the regulatory mechanism of KRP family proteins in the development of maize endosperm is still limited.Therefore,the study of the functions and regulatory roles of KRP family proteins in endosperm development,will benefit the improvement of maize grain yield and quality.In this study,the effects of ZmKRP6 and ZmKRP8 genes,which have similar gene structure and high homology,on maize endosperm cell development,grain yield and quality were studied.The results are as follows:1、zmkrp6 and zmkrp8 heterozygous Mu mutants were obtained from Maize GDB,cross-bred with wild-type inbred line W22 and generated the homozygous mutants of zmkrp6 and zmkrp8 with a single mutation site,after multi-generation backcrossing and selfing.The zmkrp6/8 double-gene mutants were obtained by cross-breeding the zmkrp6 and zmkrp8 homozygous mutant lines.The CRISPR-Cas9 knockout transgene vectors for ZmKRP6 and ZmKRP8 were constructed,transformed into maize immature embryos as explants mediated by Agrobacterium infection transformation system,to produce ZmKRP6 and ZmKRP8 knockout transgenic plants.2、The transcription level of the indicator gene,ZmE2F5,was analyzed by real-time fluorescent quantitative PCR,and was used to indirectly study the changes of ZmCDKA1 kinase activity in homozygous mutants,zmkrp6 and zmkrp8,and W22 maize inbred line.The results showed that the transcriptional level of indicator gene ZmE2F5 decreased in the mutants,indicating increased activity of ZmCDKA1 kinase.When ZmKRP6 and ZmKRP8 genes were overexpressed in leaf protoplasts,the transcription level of ZmE2F5 was up-regulated,suggests the inhibition of the ZmCDKA1 kinase activity.This results imply that ZmKRP6 and ZmKRP8 can affect the activity of ZmCDKA1 kinase.3、The developing seeds of wild-type inbred line,W22,and the two homozygous mutants,zmkrp6 and zmkrp8,were used for phenotypic analysis and cytological observation.The results showed that zmkrp6 and zmkrp8 showed significant increase in grain length,but no apparent change was observed in their grain width,during the early stages of development after pollination.Cytological observations of zmkrp6 and zmkrp8 endosperms showed increased number of cells relative to the wild-type after 5-9 days after pollination(DAP).4、In the mature seeds,zmkrp6 indicated significant increase in grain length and width,but no differences in the grain thickness relative to the wild-type.In contrast,zmkrp8 showed no significant differences in the grain length,width and thickness as compared to the wild-type.Furthermore,hundred-grain weights of zmkrp6 and zmkrp8 were considerably higher than that of the wild-type.Compared with wild-type ear,all indexes of zmkrp6 and zmkrp8 showed no significant different.5、The dynamic process of starch accumulation was analyzed in the grains of the wild-type inbred line(W22),homozygous mutants,zmkrp6 and zmkrp8,and homozygous double gene mutant,zmkrp6/8.As compared with the wild type grain,the starch accumulation rate of zmkrp6 grains was accelerated,while that of zmkrp8 grains was slowed down,but the final starch content in their endosperms remained unaffected.Similarly,the final starch content in the endosperms zmkrp6/8 mutant was not different statistically,relative to the wild-type.Although,the results of this study are still preliminary,but have proven that ZmKRP6 and ZmKRP8 can regulate the cell cycle of maize endosperm,by inhibiting the activity of ZmCDKA1 kinase.In addition,the mutation of ZmKRP6 and ZmKRP8,could promote maize grain enlargement and improve maize yield.