玉米穗长QTL EAR LENGTH7 (qEL7)的生物学功能与作用机理研究

Both ear length and kernel number per row are highly correlated with the maintenance of meristematic activity of meristems in maize inflorescence. Phytohormone biosynthesis and signaling-related genes play a central role in the regulation of the fate and meristematic activity of meristems. Early researches in our laboratory identified and isolated a QTL EAR LENGTH7 (qEL7) for ear length and kernel number per row by map-based cloning. The candidate gene of qEL7, referred to as EL7 which encodes an enzyme involving in biosynthesis of a known phytohormone, expresses in the specific domains of axillary meristems of female inflorescence. In the project, our objectives are: i) to uncover biological function of EL7 by creating transgenic maize lines with enhanced EL7 expression and with EL7 targeted CRISPR knockout; ii) to assay biochemical function of enzyme EL7 in vitro; iii) to measure hormone level in developing inflorescences of parental lines, CRISPR lines and wild type, and to identify the response of different genotypes to exogenous phytohormone; iv) to predict putative regulatory pathway of EL7 using RNA-seq data, and then knockout the key downstream genes to create single mutants and double mutants for genetic assay, take these results together, we hope to reveal the acting pathway of EL7; and v) to improve maize elite lines using marker-aided selection. The outcomes from the project will expand our understanding on the genetic control of inflorescence architecture and development, and provide the available gene for genetic improvement of ear length and kernel number per row in maize.

玉米穗长和行粒数的形成与花序分生组织的活性密切相关。植物激素参与花序分生组织活性及其命运调控。项目组前期鉴定并克隆了一个玉米穗长和行粒数相关的QTL EAR LENGTH7(qEL7)，其候选基因（暂命名为EL7）编码一个植物激素合成途径的酶，在花序腋生分生组织特异部位表达。在此基础上，本项目拟：i）通过增强EL7表达和敲除EL7，解析EL7的生物学功能；ii）采用酶动力学分析，体外证实EL7的酶学功能；iii）测定近等基因系、敲除突变体el7和野生型家系的激素水平，结合体外激素处理，鉴定不同基因型的激素水平及其对激素的响应；iv）通过RNA-seq分析、下游基因突变体和双突变体的遗传效应分析等，解析EL7的作用途径；v）应用标记辅助选择改良玉米自交系。该研究结果将丰富我们对玉米花序建成和发育调控的理论认识，也为玉米穗长和行粒数的遗传改良提供新基因资源。

在鉴定到一个控制玉米穗长、行粒数和穗重的主效QTL qEL7基础上，我们以近等基因系qEL7Ye478（长穗）和qEL7SL17（短穗）为材料，采用图位克隆技术，将qEL7定位于约50.8 kb的物理区间；其间，一个编码1-aminocyclopropane-1-carboxylate oxidase2(ACO2)的基因(ZmACO2) 的5'-UTR和上游区域在近等基因系间存在11个SNP和5个InDel。 关联分析和不同单倍型的表型分析说明，ZmACO2启动子区的变异影响其基因的表达量，是自然群体中影响穗长和行粒数变异的重要功能位点。 创制了ZmACO2的2个功能缺失突变体和3个过表达ZmACO2的转基因家系。相较于非转基因家系，ZmACO2功能缺失突变体的穗长增加0.81~0.86 cm, 行粒数增加1.80~3.49粒，穗重增加11.31~12.31 g；相反，过表达家系的穗长减少1.16~1.36 cm，行粒数减少2.06~3.76粒，穗重减少17.95~20.90 g，表明ZmACO2是一个负调控玉米穗长、行粒数和穗重的功能基因。ZmACO2在成熟叶片和幼穗中高量表达，在SPM起始原基、SPM近轴端区域、两个SM的分界区域、SM的基部以及FM处特异表达。体外酶学分析和近等基因系和转基因家系幼穗中乙烯释放测定表明， ZmACO2蛋白能够催化底物ACC产生乙烯。转录组分析发现，激素相关基因和分生组织发育相关基因在近等基因系间差异表达；内源激素测定表明，qEL7SL17幼穗中的生长素和茉莉酸的含量显著高于qEL7Ye478。我们推测乙烯可能与其它激素的互作、通过调节花序发育相关基因的表达进而调控分生组织的活性，最终引起穗部表型变异。单倍型qEL7Ye478所组配的杂交种产量显著高于qEL7SL17衍生杂交种；敲除ZmACO2功能可显著提高杂交种的籽粒产量。分子进化分析表明，ZmACO2位点在玉米驯化过程受到了选择的作用，而且优良单倍型HapYe478在现代玉米自交系中被富集。结果证实了ZmACO2的生物学功能和育种价值，首次发现了乙烯通过调节玉米分生组织活性和小花育性进而调控穗粒数形成的重要作用，为阐明玉米穗粒数形成遗传学基础积累了有益资料，也为玉米产量遗传改良提供了新基因。