基于CRISPR-Cas9定点突变改良新疆加工番茄耐贮性研究

Tomato is easy to over-ripen and softening, resulting in poor pressure resistance and lower pathogen resistance and affecting the storage and processing quality. Tomato is a respiratory climacteric fruit and produces large amounts of ethylene, namely system II ethylene, accompanied by respiratory jump. System II ethylene has autocatalytic mechanism that make it difficult to control the over-ripening process of fruits. 1-Aminocyclopropane-1-carboxylic acid (ACC) is a direct precursor for the synthesis of ethylene, ACC synthase (ACS) limits the rate of ethylene synthesis. Tomato ACC synthase genes SlACSs is a multi-gene family and the synthesis of system II ethylene involves mainly SlACS2 and SlACS4. In the study, the high specificity sgRNA would be screened from the upstream of the coding region of the SlACS2 gene by scanning the whole genome sequence of tomato for the site-specific mutation by using CRISPR-Cas9 system and no change in the sequences of SlACS4 and other SlACS genes, so as to ensure normal expression of the systym I ethylene for normal growth of plants and moderately inhibiting expression of the systym II ethylene for delaying fruit over-ripening and improving storage and transportation performance. The exogenous DNA sequences integrated on the chromosomes would be removed through self cross and genetic separation of mutants. The study would establish an efficient and safe way to breeding tomato varieties easy to storage, and provide a platform and technical support for tomato gene function study.

番茄易过熟软化，致使耐压性能变差、抗病原菌能力降低，影响贮运和加工品质。番茄为呼吸跃变型果实，伴随呼吸跃变产生大量乙烯，即系统II乙烯，其具有自催化机制，使得番茄成熟过程难以控制。1-氨基环丙烷1-羧酸ACC是合成乙烯的直接前体，ACC合酶ACS是乙烯合成限速酶。番茄ACC合酶SlACS由多基因家族编码，系统II乙烯的合成主要涉及SlACS2和SlACS4基因。通过对番茄全基因组序列扫描，从SlACS2基因上游编码区筛选出高特异性sgRNA，利用CRISPR-Cas9系统对SlACS2基因进行定点突变使其失活，同时不影响SlACS4基因及其它家族基因序列，使系统I乙烯正常表达以保证植株正常生长，又适度抑制系统II乙烯生成，迟滞果实过熟软化，提高耐贮运性能。对突变体通过自交分离剔除整合的外源DNA序列，获得安全的耐贮运新种质，为番茄新品种培育及功能基因研究提供新途径及技术支撑。

本项目以加工番茄新番72号为研究材料,建立优化了遗传转化体系,克隆了SlACS2基因并基于CRISPR/Cas9系统对该基因进行了定点编辑，具体研究结果如下：克隆新番72号的SLACS2基因长度1458bp，以此作Querry，在番茄的基因组数据库进行Blast比对，结果表明该基因位于番茄的1号染色体，由4个外显子、3个内含子组成；在SlACS2的第2外显子区域设计2个靶位点，构建双靶点的CRISPR/Cas9 敲除载体并导入农杆菌；以OD600=0.7的菌液侵染子叶15min，培养后检测到抗性愈伤诱导率为54.6%，抗性不定芽分化率为24.67%，获得再生植株78株；进行PCR检测得到57株cas9阳性植株，其中目标基因突变株9株，包含纯合突变2株；将T0代植株移至温室自花授粉，从分离后代中筛选出无T-DNA的SlACS2 突变株6个，均为缺失突变，即在gRNA2位点缺失了ccaactt7个碱基；对T2和部分T3代的1063个单株进行测序，归纳出21种不同编辑类型，主要以小片段或单碱基的缺失插入为主，亦有少量的碱基替换，但未发现有两个sgRNA之间的大片段的缺失；从中筛选出能够稳定遗传无T-DNA组份的典型编辑类型3种，以野生型新番72号为对照，分别选取3个单株的四个生理时期进行ACC含量、ACS酶活性及乙烯释放量等测定，发现3种编辑类型的四个不同时期的ACC含量、ACS酶活性以及乙烯释放量均明显低于对照；测定红熟期的果实硬度、耐压力及贮藏损失率等，结果最大较对照的果实硬度提高6.6%，耐压力提高9.8%，损失率减少13.9%。上述研究结果初步说明，对番茄SlACS2基因进行定点编辑，可降低ACS酶活性，适度抑制系统II乙烯生成，达到迟滞果实过熟软化，提高耐贮运性能的效果，为创制番茄耐贮运新种质做了有益探索。