黄瓜软果刺性状遗传调控机制研究

Generally, cucumber fruit spines of Northern Chinese type in China are hard and feel prickly, and they go against manual picking and are hard for washing and eating, which shows a significant influence to consumer. Therefore, it is significant to study genetic modulation of tender fruit spines and new variety breeding of tender fruit spines in cucumber. A mutunt of tender fruit spines of was used in this study, and genetic analysis in early study proved that one single recesive nuclear gene, ts (tender spines), determines the tender fruit spines trait in cucumber. For the tender fruit spines trait, the large F2 population and map-based cloning technology will be used for isolating the ts gene. Co-segregation analysis and complementation test of genetic transformation in cucumber will reveal the function of the ts gene. Then a series of experimental means, such as homologous analysis, subcellular localization, quantitative RT-PCR, mRNA in situ hybridization, would be performed to understand the function of the ts gene. Moreover, many other experimental technologies,such as preparation of ts polyclonal antibody, chromatin Immunoprecipitation (ChIP) and ChIP-Seq, yeast one-hybrid, transcriptome analysis, electrophoretic mobility shift assay, transcriptome analysis, would be performed to obtain target genes of ts and analyse genetic regulatory networks of ts and its target genes, to reveal the genetic mechanism of the tender fruit spines. For the ts gene, easy-testing markers would be developed for marker-assisted selection of tender fruit spines trait in cucumber breeding.

国内普遍种植的华北型黄瓜均为硬果刺，坚硬扎手，不利于采摘、清洗与食用。因此，研究黄瓜软果刺性状及选育软果刺品种具有重要的意义。本项目利用一个黄瓜软果刺突变体（tender spines，ts）为材料，前期确定该性状是核单基因控制的隐性性状。本项目针对软果刺性状，利用F2大群体，采用图位克隆技术分离ts，并进行共分离分析和黄瓜遗传转化验证，通过同源分析、亚细胞定位、定量RT-PCR、mRNA原位杂交等多种实验对ts进行功能分析。同时，制备ts蛋白抗体，进行染色质免疫共沉淀（ChIP）及ChIP-Seq、酵母单杂交及凝胶阻滞(EMSA)实验，并结合转录组数据筛选ts基因直接调控的下游靶基因，进一步分析ts基因及靶基因的功能及所参与的调控网络，揭示黄瓜软果刺性状形成的遗传机理。同时，本项目将ts开发成易于检测的分子标记，用于黄瓜软果刺性状分子标记选择育种。

黄瓜属于葫芦科家族，是世界上非常重要蔬菜作物之一。果刺和表皮毛在植株适应周围环境、抵抗外界损伤、抵御病原菌侵染和防止昆虫的啃噬等方面，起着非常重要的作用。.本项目的实验材料为黄瓜野生型和软刺突变体（tender spines，ts）。扫描电镜结果显示野生型的果刺由一个圆锥状的顶细胞、数个茎细胞和多细胞组成的膨大球状刺座组成。而软刺突变体ts的果刺由一个圆锥状的顶细胞、2~3个茎细胞以及多个细胞不规则排列形成的棒球棒状刺座组成。.软刺性状的遗传分析表明，软刺是由细胞核单基因控制的隐性性状。利用软刺突变体材料NC073和不同野生型材料构建群体，利用高密度SSR遗传图谱，将软刺基因ts定位到第一号染色体的109.7kb的区域内。在该区域内有15个基因，基因序列比对发现，Csa1G056960基因的第二个内含子剪切位点由GT变为GC，导致编码区第二个外显子缺失。该基因编码C-型植物凝集素络氨酸受体激酶，突变后N端缺少27个氨基酸，从而使硬刺突变为软刺。.组织特异性表达实验证明基因Csa1G056960在果刺部位特异表达，该基因在无毛突变体tril和微表皮毛突变体mict中的表达量大幅下调。烟草瞬时表达实验显示Csa1G056960定位到细胞膜上。mRNA原位杂交实验进一步证明Csa1G056960基因特异表达于刺座部位。分别测定了野生型和软刺突变体ts果刺中木质素的含量，结果表明Ts基因突变后可能促进木质素的合成。此外，统计结果发现，Ts基因的突变会使果刺及表皮毛的密度增大。.将野生型、软刺突变体ts、无毛突变体tril和微毛突变体mict的幼果进行转录组测序并比对分析，发现与木质素合成、响应植物激素信号的基因，在野生型和不同突变体材料中的表达量有明显的差异。对这些基因进行RT-PCR表达验证和功能分析，证明这些基因很可能参与了黄瓜果刺发育调控过程。