烟草“8306”NtCPS2基因敲除及叶面化学成分改良

Leaf surface chemical component improvement by NtCPS2 gene knock out in tobacco line “8306”

8306是重要的高香气烤烟育种材料,因其腺毛分泌物中含有赖百当化合物而使其杂交后代具有晾晒烟香气特征。为此,本研究利用CRISPR/Cas9技术,对8306中赖百当生物合成途径的关键酶基因NtCPS2进行敲除,获得了gRNA区发生单碱基插入突变导致翻译提前终止的纯合突变体8306-1。与8306相比,该突变株系生长发育正常,腺毛类型和腺毛密度无显著变化;利用GC-MS技术对叶面化学成分进行检测,在8306-1中并未发现8306中所含有的顺冷杉醇和赖百当二醇等赖百当类化合物,而腺毛分泌物的其他主要成分,如西柏三烯一醇、西柏三烯二醇及蔗糖酯的含量与对照相比并无明显变化;RT-PCR检测分析显示,8306-1与8306中MEP途径及二萜生物合成途径的关键基因,如NtDXS、NtDXR、NtCMS、NtGPPS、NtGGPPS、NtCYC、NtCYP71D16等在转录水平上的表达均无明显差异。该研究结果表明,NtCPS2基因敲除能有效降低烟草赖百当类二萜化合物的含量,而对叶面其他化学成分及类萜途径基因表达无明显影响。因此,NtCPS2基因是进行烟草叶面化学成分定向改良的理想靶点,对烤烟高香气育种具有重要意义。

Tobacco line 8306 is an important breeding germplasm for flue-cured tobacco with high aroma content.However,due to the existence of labdanoids in its glandular trichome exudates,the sun-cured tobacco scent is mixed in its smoke,which limits its application.Thus,NtCPS2,the key gene of labdanoids biosynthesis was knocked out using CRISPR/Cas9 technology in this study and a mutant,named 8306-1,was obtained.8306-1 carries a single nucleotide insertion in the gRNA region,which resultes a premature stop codon and function loss of NtCPS2.Compared with 8306,8306-1 showed normal growth and development,and no significant changes of trichome type and density were found.GC-MS analysis showed that no Z-abienol and labdene-diol were detected in 8306-1 glandular trichome exudates,while no significant changes were observed for the content of other components such as cembratrien-ol,cembratrien-diol and sucrose ester as compared with the control.RT-PCR analysis showed that there was no significant difference in the expression of MEP pathway genes such as NtDXS,NtDXR,NtCMS,and cembranoids biosynthesis pathway gene NtCYC and NtCYP71D16 between 8306-1 and 8306.Our results showed that knock out of NtCPS2 gene can effectively down-regulate the biosynthesis of labdanoids,but has no obvious influence on other chemical components of leaf surface.Therefore,NtCPS2 is an ideal target gene for improvement of tobacco leaf chemical components and high aroma flue-cured tobacco breeding.