| Maize(Zea mays L.)is the most widely produced and consumed cereals,and used as human food,animal feed and industrial raw material for starch,oil,and other products.With a continuously growing population,ensuring continuous increases in maize yields is becoming increasingly important.Increasing the yield of maize and improving the quality(i.e.,amino acid content,lysine and tryptophan content,and starch/amylose/amylopectin content)have always been the focus of crop science.In the past decades,factors like plant hormones,sugars,peptides/receptors,and transcription factors were identified in early maize kernel development.However,the mechanisms are largely unknown in the filling and dehydration stages.It will be interesting and meaningful to understand the endosperm development and molecular regulatory networks for the plant scientists and breeders.In this study,a natural variation mutant that causes abnormal kernel development,and displays a shrunken kernel phenotype,was identified and named "shrunken 2008(sh2008)".Genetic analysis showed that the phenotype was a recessive trait controlled by a mononuclear gene.The target gene ZmThx20 was located by map-based cloning,and its function was analyzed.This study plays a role in enriching the understanding of endosperm development and molecular regulatory networks.1.Phenotypic identification of mutant sh2008The mutants seeds can be clearly distinguished at DAP(days after pollination)12,and continued appear milky white.With the increase of pollination days,the gradually became shriveled and shrunk after full maturity.The 100 seed weight of sh2008 was only 30%of the wild type.The seed germination test showed that most mutant seeds could not germinate.The DAP20 seed embryos were culture on 1/2 MS medium.We found that the germination rate of mutant embryos was not different from that of WT(wild type).The seedlings grow weaker after transplanting,and can blossom,pollinate and bear fruit normally under normal management conditions.The total starch content of mutant grain was only 26%of that of WT,and the total storage protein was about 70%of that of wild type control.SDS-PAGE showed that the content of zein decreased,especially 19kDa and 22kDa α-zein.The soluble sugar content in mutant grains maintained a high level during the whole grain filling development,and the content of free amino acids changed significantly.The paraffin section showed that the accumulation of starch particles was less and the area of the basal endosperm transfer cell layer was smaller in the development of mutant seeds.The scanning electron microscope was used to further observe the natural section of mature seeds.We found that the starch particles in mutant seeds were relatively loose,with less surrounding protein matrix,and there were many irregular holes on the surface of starch particles.Therefore,gene mutation seriously affected the development of endosperm in maize grains.2.Genetic analysis of mutantsThe sh2008/+maize line was crossed with the inbred lines B73,Q319,and W22 to produce the segregation populations with different genetic backgrounds.Chi-square test of the F2 kernels(normal:the shrunken=3:1)from the segregation populations showed that the sh2008 allele is a monogenic recessive nuclear mutation.3.Map-based cloning and genetic complementarity of mutantsTaking sh2008/B73 F2 population as the research object,the gene was located on chromosome 5(178-208M)by bulked-segregant analysis.Further develop molecular markers and finally lock the gene between markers m190-2 and m190-6.The candidate genes in this interval were analyzed and their expression was semi-quantitatively analyzed.All candidate gene fragments of WT and sh2008 were amplified by PCR.The sequencing results showed that there were multiple insertion deletion fragments in the second exon of ZmThx20(GRMZM2G169580),in which the deletion of T base(+2246bp)led to the emergence of the stop codon in translation,while other candidate genes did not change.Therefore,Zm Thx20 was identified as the candidate gene for the sh2008 locus.The transgenic complementation test showed that the overexpressed gene ZmThx20 could restore the phenotype.Wild-type calli were transformed using the CRISPR/cas9 editing system,and the offspring of transgenic events showed the same shrunken phenotype.4.Functional analysis of Zm Thx20ZmThx20 is a member of plant specific transcription factor Trihelix family.Evolutionary tree analysis shows that it belongs to the GT-2 subfamily of the Trihelix family.RT-PCR analysis showed that the expression of ZmThx20 was detected in different organs and kernels at DAP8,and its expression in kernels continued later than DAP30,with the maximum level at DAP 16.ZmThx20 expression during kernel development is more modest than in roots,stems,or leaves.Subcellular localization showed that Zm Thx20 was only located in the nucleus.The full length of transcription factor ZmThx20 had no self-activation activity,but its N-terminal had self-activation activity.Several genes were screened by yeast two-hybrid,but most of them were located in the cytoplasm and only a few in the nucleus.Luciferase complementation test showed that ZmThx39 could interact with it.Yeast single hybrid and bimolecular fluorescence complementation tests showed that the ZmThx20 could bind to the promoter region of the 19kDa zein gene and significantly improve the transcriptional activity.5.Gene mutation leads to disorder of starch synthesis and metabolismTranscriptome sequencing of sh2008 mutant grains 15 days after pollination showed that gene mutation led to many gene up regulation and down regulation.GO enrichment analysis showed that most of the DEGs enriched in GO terms of translation,ribosome and nutrient reservoir activity and development.Further detection of starch synthesis and protein regulatory gene expression levels found that most of the genes changed,lead to defects in endosperm development and seed storage. |
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