高烟碱K326定向改良材料的创制与分析  

Creation and analysis of high nicotine oriented modified material K326

作　　者：郑淑心 闫筱筱 王召军 张洪映[1] 崔红[1] ZHENG Shuxin;YAN Xiaoxiao;WANG Zhaojun;ZHANG Hongying;CUI Hong(College of Tobacco Science,Henan Agricultural University,Zhengzhou 450002,China)

机构地区：[1]河南农业大学烟草学院,郑州450002

出　　处：《烟草科技》2022年第7期1-8,共8页Tobacco Science & Technology

基　　金：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)“基于CRISPR/Cas9技术创制高生物碱材料及蚜虫抗性鉴定”(212102110046);河南省自然科学基金项目“MeJA诱导烟草分泌型腺毛发生关键调控基因的筛选和功能研究”(182300410094)。

摘　　要：为了明确NtJAZ1基因对烟碱含量(质量分数)和烟株生长发育及品质的影响,以烤烟品种K326为材料,利用CRISPR/Cas9技术对NtJAZ1基因进行敲除,获得了纯合突变株系NK-9。测序结果表明,NtJAZ1-1在1587~1641bp之间大片段缺失,NtJAZ1-2在1547~1548bp之间缺失了2个碱基,导致翻译提前终止。RT-PCR检测结果表明,NK-9烟碱生物合成关键基因NtMYC2a、NtMYC2b、NtPMT和NtQPT的表达水平显著提高。田间调查发现,NK-9株系生长发育正常,株高、叶数和叶宽显著高于对照K326,但叶长显著低于K326。显微观察表明,NK-9叶面腺毛密度较对照有所降低。对现蕾期、打顶期和成熟期烟碱含量检测发现,NK-9株系中部叶的烟碱含量分别比对照提高33.26%、36.41%和39.28%。烤后烟叶化学成分分析表明,NK-9常规化学成分比例协调,烟碱含量较对照提高44.91%;中性香气成分中新植二烯、类胡萝卜素类降解产物含量明显增加,但棕色化反应产物、苯丙氨酸类降解产物和茄酮含量有所降低。说明敲除NtJAZ1基因能够有效提高烟叶烟碱含量,对腺毛发育和叶面化学成分的积累也有一定影响。In order to clarify the effects of NtJAZ1 gene on nicotine content(mass fraction),growth and development,and quality of tobacco plants,a homozygous mutant NK-9 was obtained by CRISPR/Cas9 technology through the knockout of NtJAZ1 gene in flue-cured tobacco cultivar K326.The sequencing results indicated that the bases of NtJAZ1-1 located between 1587-1641 bp and two bases of NtJAZ1-2 located between 1547-1548 bp were lost,which generated the premature termination of gene translation.RT-PCR results showed that the expression levels of the key genes,NtMYC2a,NtMYC2b,NtPMT,and NtQPT,in nicotine biosynthesis process increased significantly.The field experiment tests revealed that NK-9 line grew and developed normally,and its plant height,leaf number and leaf width were significantly higher than those of the control(K326),while its leaf length was significantly lower than that of K326.The density of trichome in leaf surface of NK-9 was lower than that of K326 indicated by microexamination.The nicotine content in the middle leaves of NK-9 at flower-bud appearing,topping and mature stages increased by 33.26%,36.41%,and 39.28%respectively compared with K326.Chemical composition analysis of the cured leaves showed that NK-9 had the balanced chemical compositions and a 44.91%higher content of nicotine than those of K326.The contents of neophytadiene and carotenoid catabolites of neutral aroma components increased significantly,while the contents of browning reaction,phenylalanine catabolites and solanone decreased.All results indicated that NtJAZ1 gene knockout effectively increased the contents of alkaloids in NK-9 and had certain effects on the trichome development and the accumulation of leaf surface chemical components.

关 键 词：烟草 基因编辑 NtJAZ1 烟碱 生长发育 

分 类 号：S572[农业科学—烟草工业]