辣椒突变系E29矮秆基因的克隆与功能鉴定

Dwarf plants are characterized in strong anti-lodging ability and high group photosynthetic efficiency, thus they fit for high density plantings and mechanized cultivation management. Breeding the dwarf varieties is one important aspect of pepper breeding. Dwarf genes are highly valuable for breeding dwarf varieties in pepper. However, currently there hasn’t been any clone of dwarf genes. In this study, a novel dwarf mutant line, which could control the plant height of pepper, was discovered by EMS (ethyl methane sulfonate) mutation and designated as a single recessive gene. Then two candidate dwarf genes and two SNPs loci were located by whole-genome sequencing. Based on these findings, we plan to clone the dwarf gene through SNP markers test in F2 segregating and expression of gene transcription level analysis with qRT-PCR test. At last, over-expression and VIGS(virus induced gene silencing) technologies are employed to identify the dwarf gene’ s function. This study will not only provide new gene resource for breeding dwarf varieties, but also provide new insight into the understanding of the molecular mechanisms of dwarf character.

矮秆品种抗倒伏能力强、群体光合效能利用率高，适于高密度种植和机械化栽培管理。选育矮秆品种是辣椒育种的一个重要方向。矮杆基因在高效选育辣椒矮杆品种中具有重要的利用价值，然而目前仍无辣椒矮秆基因被克隆。本研究前期采用EMS诱变获得了一份辣椒矮杆突变系E29，遗传分析表明矮秆性状受单隐性基因控制；全基因组混池测序定位到2个矮秆候选基因并开发了2个SNP标记位点。拟在此基础上利用已开发的SNP标记对扩大的F2群体进行验证，初步确定矮秆基因；再采用实时荧光定量PCR法分析基因在转录水平上的表达量进一步确定矮秆基因；最后采用基因过表达技术和基因沉默技术鉴定矮秆基因的功能。研究结果不仅为辣椒矮杆育种提供新基因资源，还能为进一步深入了解辣椒矮秆性状分子机理奠定基础。

品种矮化是辣椒育种发展趋势。创制矮秆辣椒新种质、发掘控制矮秆性状的新基因或等位基因有重要的实际与科学价值。前期研究中，从甲基磺酸乙酯（EMS）诱变群体中鉴定出的一株矮化的辣椒突变体，是用于选育矮化新品种的优良亲本材料。.E29为BR不敏感突变体。遗传分析表明，矮化突变受单个隐性基因控制。全基因组重测序，dCAPs分析证实CaBRI1为矮化突变候选基因。6421和E29同一器官中CaBRI1表达量基本相同。说明对于EMS诱变产生的点突变，不能采用通过测定突变基因表达量方式验证候选基因。CaBRI1中非同义单核苷酸突变（C to T）导致CaBRI1激酶结构域（S_TKc结构域）第1157个氨基酸由Pro突变成Ser（Pro1157Ser），激酶活力检测发现Pro1157Ser损害了激酶活性。亚细胞定位发现Pro1157Ser不能使该蛋白从细胞膜上掉落。构建CaBRI1基因沉默载体，沉默6421、E29及辣椒易感材料茄门，实验结果表明证实CaBRI1为控制株高性状的基因。6421和E29转录组测序将差异表达基因富集到BR合成途径上，BR合成关键基因CaDWF4、CaROT3在E29中上调，BL含量在E29中也显著升高，说明CaBRI1点突变影响了E29中活性BR的积累，从侧面验证了CaBRI1为控制突变性状的基因。.建立了6421、E29子叶外植体离体再生体系。以MS为基本培养基，1.0 mg/L 6-BA 和0.5 mg/L IAA适宜用作诱导愈伤组织产生，0.1 mg/L 2,4-D有利于愈伤组织增殖，9.0 mg/L 6-BA适宜用作不定芽诱导，3.0 mg/L 6-BA，0.3 mg/L GA3和0.1 mg/L IAA适宜用作芽伸长。伸长的芽直接转接至不添加生长调节剂的1/2 MS培养基中，生根率100%。构建了CaBRI1过表达载体转化E29子叶外植体，优化了E29遗传转化体系。潮霉素B适宜的筛选浓度为20 mg/L，特美汀最适宜浓度为300 mg/L，共培养最适时间为3 d。获得了大量抗性愈伤组织，但目前还未获得阳性植株。.该研究发现了一个辣椒株高控制基因CaBRI1新等位变异，对培育适宜设施栽培与机械化收获辣椒品种具有理论与应用价值。E29遗传转化体系实验为今后辣椒遗传转化效率的进一步提高及CaBRI1基因功能鉴定提供基础。