调控玉米叶表皮形态建成的SEP1和DSP1基因克隆与功能分析

Stomata and epidermal cell, two major type epidermis, play important roles in regulating photosynthesis, water use efficiency and heat dissipation etc. As the morphology is clear and easy to observe, maize (Zea mays. L) leaf epidermis is an excellent material to study the epidermal morphology of monocotyledonous plants. At present, the molecular mechanism of regulating stomata and epidermal cell growth and morphogenesis in maize is still not clear. Early research in the laboratory, two mutants sep-1 (smooth epidermal pavement cell lobes -1), showing epidermal pavement cells with smooth marginal lobes, and dsp-1 (disturbed stomatal pattern -1), showing stomata clustering and abnormal stomata, were screened. Their phenotypes are regulated by a single gene recessive inheritance. In this project, we intend to clone SEP1 and DSP1 candidate genes by constructing large BC2F2 separation groups and combining with genomic high-throughput sequencing technology; to obtain the allelic mutants for allelic detection via genetic transformation; to elucidate the biological function of candidate genes and develop functional markers; The expression of SEP1 and DSP1 in the positive and negative single strains of genetic transformation were analyzed and the tissue parts of each gene expression were clearly determined. It is preliminarily illuminated that SEP1 and DSP1 regulate the mechanism of corn epidermal morphogenesis. The conclusion of this study is helpful to further perfect the mechanism of stomatal and epidermal cell development in maize, and it provides beneficial genetic resources to improve the genetic improvement of adversity stress and yield traits of crops.

气孔及表皮细胞在调节植物光合作用、水分利用及热量散失等方面发挥重要作用。玉米叶表皮形态清晰，便于显微观察，是深入研究单子叶植物表皮形态建成的极好材料。目前，玉米中调控表皮生长发育与形态建成的分子机制仍不清楚。课题组前期工作中分离到两个表皮变异单基因隐性遗传突变体sep-1和dsp-1，分别表现出平滑的扁平细胞边缘凸出和多个气孔簇生的表型；本项目拟通过构建大的BC2F2分离群体，结合基因组高通量测序技术，克隆SEP1和DSP1候选基因；获得等位突变体进行等位性检测及遗传转化验证，阐明候选基因的生物学功能并开发功能标记；对遗传转化后代阳性及阴性单株中SEP1和DSP1进行表达分析，明确各自基因表达的组织部位；初步探明SEP1和DSP1调控玉米表皮形态建成信号转导机制。该项目的研究结果有利于进一步完善玉米表皮形态建成的机制，且为提高农作物的逆境胁迫和产量性状的遗传改良提供有益的基因资源。

本研究通过正向遗传学手段分离到玉米叶表皮细胞边缘凸出变平滑突变体sep-1(smooth epidermal pavement cell lobes -1)，对干旱胁迫更敏感，与B73杂交，构建BC2F2分离群体，结合基因组高通量测序，转录组测序和图位克隆技术定位到SEP1候选基因，位于第7染色体Bin 7.06位置。精细定位候选基因到160 kb，对区间内12个候选基因测序，发现编号GRMZM2G342386基因CDS第949位置碱基由G变T，导致第317位置氨基酸由E（谷氨酸）变成终止密码子，蛋白翻译提前终止。在sep-1中，ZmARP2基因表达极显著降低，初步确定为候选基因。通过同源性比对和生物信息学分析发现，该基因与拟南芥中AtARP2（Actin related protein 2）高度同源，是玉米中的微丝合成相关基因，属于ARP2/3复合体家族，调控玉米表皮细胞形态建成。同时我们利用反向遗传学手段，分离到2个控制气孔发育的单基因隐性遗传突变体Zmice1-1 (inducer of cbf expression1-1)和Zmice2-1，与对照相比，Zmice1-1叶片气孔密度和气孔指数极显著降低，打破了一个气孔间隔一个表皮长细胞的排列模式；Zmice2-1叶气孔密度与对照无显著性差异。利用CRISPR–Cas9基因编辑技术获得不同位点等位突变体Zmice1-2等，对其表型鉴定发现，Zmice1-2等都有气孔异常表型，并且和Zmice1-1有类似的气孔表型，证明了ZmICE1的确参与气孔发育的调控。此外，我们利用理化诱变手段，诱变玉米自交系B73、K22、LY8405、郑58和PH6WC，创制了超过2000个系的突变体库，筛选到株型、叶型、叶色、穗型、粒型、生育期、育性、根系等不同农艺性状相关突变体80余个，为玉米遗传育种创制了丰富的种质资源。