高分泌型无赖百当烟草8306突变材料的创制与分析  

Creation and analysis of highly secretory and labdane free tobacco 8306 mutant material

作　　者：李旖梅 王召军 闫筱筱 张洪映[1] 任书乐 李雪君[2] 孙计平[2] 崔红[1] LI Yimei;WANG Zhaojun;YAN Xiaoxiao;ZHANG Hongying;REN Shule;LI Xuejun;SUN Jiping;CUI Hong(College of Tobacco Science,Henan Agricultural University,Zhengzhou 450002,China;Tobacco Research Institute,Henan Academy of Agricultural Sciences,Xuchang 461000,Henan,China)

机构地区：[1]河南农业大学烟草学院,郑州市450002 [2]河南省农业科学院烟草研究所,河南省许昌市461000

出　　处：《烟草科技》2022年第4期1-8,共8页Tobacco Science & Technology

基　　金：国家烟草专卖局基因组重大专项项目“烟草腺毛功能基因挖掘与分子调控研究”(110201401003);国家自然科学基金项目“中国野生毛葡萄VqJAZ4抗白粉病功能及调控机制研究”(32102361);河南省重点研发与推广专项(科技攻关)项目“基于CRISPR/Cas9技术创制高生物碱材料及蚜虫抗性鉴定”(212102110046)。

摘　　要：8306是具有晾晒烟风格的烤烟育种材料。在前期试验中创制了NtCPS2基因突变株系8306-1,抑制了晾晒烟风格代表性物质赖百当二萜的合成。为进一步提高8306-1株系中西柏烷二萜和蔗糖酯含量(质量分数),采用基因编辑技术对8306-1株系的NtCycB2基因进行敲除,获得了纯合突变株系8306-2。与8306、8306-1株系相比,8306-2株系生长发育正常,农艺性状无显著差异。8306-2株系中长柄分泌型腺毛密度显著高于8306和8306-1株系,且存在较多的分枝型腺毛。8306-2与8306-1株系腺毛分泌物组成成分相同,均缺少8306中的顺冷杉醇和赖百当二醇等赖百当类化合物;且8306-2株系叶面化学成分含量(质量分数)比8306和8306-1株系显著提高,其中西柏烷二萜含量分别提高95.7%和93.4%,蔗糖酯含量分别提高60.8%和62.5%。qRT-PCR检测分析发现,8306-2株系中NtCBT、NtCYP71D16、NtCPS2和NtABS 4个二萜合成关键酶基因的表达水平显著高于8306-1和8306株系,而二萜合成的上游基因NtGPPS和NtGGPPS的表达无显著变化。表明8306株系中NtCycB2基因和NtCPS2基因的共敲除,在有效抑制赖百当类物质合成的基础上显著增加了烟草腺毛密度和腺毛分泌物含量。8306 Line is a flue-cured tobacco breeding material with the flavors of air-cured and sun-cured characters.In previous studies,an NtCPS2 gene mutant line,named as 8306-1,was created to inhibit the synthesis of labdane diterpenoids,the representative flavor substances of air-cured and sun-cured tobacco.To further increase the mass fraction of cembranoid diterpene and sucrose ester in8306-1 line,gene editing technology was used to knock out the NtCycB2 gene of 8306-1 line,and a homozygous mutant strain 8306-2 was obtained.Compared to 8306 and 8306-1,the growth and development of 8306-2 line was normal,and there were no significant differences in agronomic traits.The density of long stalk secretory glandular trichomes of 8306-2 line was significantly higher than those of 8306 and 8306-1 lines,and 8306-2 line had more branched glandular trichomes.The compositions of glandular trichome secretions of 8306-2 and 8306-1 lines were the same,which both lacked the cis-abienol and labdenediol in 8306.Meanwhile,the mass fraction of leaf surface chemistry of 8306-2 line were higher than those of 8306 and 8306-1 lines,among which the content of cembranoid diterpene increased by 95.7%and 93.4%respectively,and the content of sucrose ester increased by 60.8%and 62.5%respectively.qRT-PCR analysis found that the expression levels of the four key diterpene synthesis enzyme genes NtCBT,NtCYP71D16,NtCPS2 and NtABS of 8306-2 line were significantly higher than those of 8306-1 and 8306 lines,while there was no significant difference in the expressions of the upstream genes NtGPPS and NtGGPPS for diterpene synthesis.It indicated that the co-knockout of NtCycB2 gene and NtCPS2 gene of 8306 line significantly increased the density and secretion of tobacco glandular trichomes on the basis of effectively inhibiting the synthesis of labdane.

关 键 词：烟草 8306株系 分泌型腺毛 NtCycB2 基因敲除 

分 类 号：TS413[农业科学—烟草工业]