【作者】 张鹏；

【导师】 吴承来；

【作者基本信息】 山东农业大学 ， 作物学， 2022， 硕士

【摘要】 玉米是重要粮饲兼用作物,其籽粒大小是影响产量的重要因素。玉米籽粒属于胚乳型种子,淀粉占胚乳重量的90%,淀粉的充实程度直接影响籽粒的重量。因此,研究玉米籽粒发育对提高玉米产量和指导品种选育具有重要意义。本研究对课题组在育种过程中获得一个玉米籽粒自然突变体suk1（sunken kernel 1）进行表型分析、生理生化分析、遗传分析、组织切片观察、基因定位以及对突变基因进行表达分析等工作,主要结果如下:（1）突变体性状分析:suk1和野生型籽粒在授粉后20 d开始出现差异,表现为suk1籽粒顶部膨大饱满,野生型籽粒顶部开始凹陷,而suk1成熟籽粒表现为顶部深度凹陷,野生型籽粒凹陷程度较浅。suk1的胚和胚乳都能正常发育,但与野生型相比百粒重低15.8%,发芽率降低5%,suk1幼苗稍弱,而成熟期植株表型与野生型无差异。suk1籽粒鲜重和体积在授粉后20 d、25 d和30 d显著高于野生型,而在授粉后45 d显著低于野生型;籽粒干重授粉后各时期均显著低于野生型。suk1成熟籽粒中胚乳细胞小于野生型,淀粉粒表面皱缩凹陷。淀粉形成和积累受阻,淀粉含量显著降低。（2）突变基因定位与变异分析:构建suk1与B73、W22和Mo17三种不同遗传背景的F₂群体,通过遗传规律分析,证明suk1的表型受隐性单基因控制。用B73、suk1和F₂隐性池进行BSA测序分析,将突变基因初步定位在9号染色体0～30 Mb之间,开发Indel标记进一步精细定位,将突变基因定位在标记In10.86和In10.96之间100 Kb范围内,其中包括5个候选基因。通过候选基因功能分析、RNA-seq结果分析、q RT-PCR结果分析和等位基因验证,证明Zm00001d045042为目的基因,并且突变基因为Sh1的新等位基因。突变发生在Zm00001d045042第8外显子的4个碱基（ATTC）缺失,导致终止密码子提前出现,最终使蔗糖合成酶活性降低。（3）淀粉合成基因表达分析:通过对野生型和suk1授粉后不同天数的籽粒中Mn1、Sus1、Su1、Sbe3、Bt2和Sh2的表达水平进行q RT-PCR分析,suk1籽粒中Mn1,Sus1、Su1、Sbe3、Bt2和Sh2的表达水平都显著提高。Sh1突变导致蔗糖合成酶活性降低,蔗糖含量升高,影响了淀粉的合成,但与淀粉合成相关基因的上调表达,弥补了一部分Sh1突变引起的淀粉合成缺陷。更多还原

【Abstract】 Maize is an important food and feed crop,and its kernel size is an important factor affecting the yield.Maize kernels are endosperm-type seeds,and starch accounts for 90%of the weight of the endosperm.The degree of starch filling directly affects the weight of the kernels.In this study,a natural mutant of maize kernel suk1（sunken kernel 1）obtained by the research group during the breeding process was subjected to phenotypic analysis,physiological and biochemical analysis,genetic analysis,tissue section observation,gene mapping,and expression analysis of mutant genes.The main results are as follows:（1）Trait analysis of mutants:The difference between suk1 and wild-type kernels began to appear at 20 d after pollination.The tops of suk1 kernels were swollen and plump,and the tops of wild-type kernels began to sag,while suk1 matured kernels showed deep sag at the top,and wild-type kernels had a shallower sag.Both the embryo and endosperm of suk1 developed normally,but compared with the wild type,the 100-kernel weight was 15.8%lower and the germination rate was 5%lower.The fresh kernel weight and volume of suk1 were significantly higher than those of the wild type at 20 d,25 d and 30 d after pollination,but significantly lower than those of the wild type at 45 d after pollination.Endosperm cells in mature kernels of suk1were smaller than those of wild type,and the surface of starch kernels was shriveled and concave.Starch formation and accumulation were hindered,and starch content was significantly reduced.（2）Mutant gene mapping and variant analysis:F₂ populations with three different genetic backgrounds of suk1 and B73,W22 and Mo17 were constructed,and the phenotype of suk1was controlled by a single recessive gene through genetic analysis.B73,suk1 and F₂ recessive pools were used for BSA sequencing analysis,and the mutant gene was initially located between 0～30 Mb of chromosome 9,and the Indel marker was developed for further fine mapping.The mutant gene was located between the markers In10.86 and In10.96 100 Kb range,including 5 candidate genes.Through candidate gene function analysis,results and analysis of RNA-seq,results and analysis of q RT-PCR and allele verification,it was proved that Zm00001d045042 was the target gene and the mutated gene was a new allele of Sh1.The mutation occurred in the deletion of 4 bases（ATTC）in exon 8 of Zm00001d045042,which resulted in the premature appearance of the stop codon and ultimately reduced sucrose synthase activity.（3）Expression levels of Mn1,Sus1,Su1,Sbe3,Bt2 and Sh2 in suk1 kernels by q RT-PCR analysis of the expression levels of Mn1,Sus1,Su1,Sbe3,Bt2 and Sh2 in wild-type and suk1kernels at different days after pollination were significantly increased.Sh1 mutation leads to the decrease of sucrose synthase activity and the increase of sucrose content,which affects starch synthesis.However,the up-regulated expression of genes related to starch synthesis compensates for some of the starch synthesis defects caused by Sh1 mutation.更多还原