J Exp Bot:农科院烟草所张洪博研究组发表烟碱代谢关联基因挖掘研
近日，国际植物学知名刊物《Journal of Experimental Botany》上在线发表了中国农业科学院烟草研究所烟草功能成分与综合利用创新团队在烟草烟碱代谢关联基因挖掘研究方面取得进展。研究生马浩然为第一作者，张洪博研究员为通讯作者。
烟碱(又名尼古丁)是烟草叶片中的主要生物碱，约占叶片干重的0.6%–3.0%，烟碱含量是烟草和烟草制品的关键质量指标。研究证明，烟碱在烟草根部合成，经木质部输送至烟叶及其他地上部器官，其合成与烟草根系中的基因表达调控密切相关。尽管已有多个烟碱代谢相关基因得到克隆，但其调控机制仍不明确，限制了分子技术在烟草品质育种中的应用。
上世纪30年代以来，科学家利用Burley 21烟草的低烟碱突变体nic1和nic2进行了大量烟碱代谢调控研究工作，先后鉴定了若干烟碱合成相关标记基因，并克隆了nic2突变体基因，但nic1突变体基因还未得到鉴定。本研究通过分析低烟碱突变体根系中的基因表达特性，新发现了碱性几丁质酶基因PR3b在Burley 21烟草中的转录后可变剪切现象，分析了该现象在低烟碱突变体和对照烟草间的生物学差异，并研究了其对PR3b的蛋白结构和特异酶活性影响。该研究还利用遗传学方法证明了PR3b可变剪切与低烟碱突变体nic1和nic2之间的分子关联。后续，该团队将分离操纵PR3b可变剪切的生物酶基因，并进一步克隆nic1突变体基因，以揭示烟草的烟碱代谢调控机制。
原文链接：
Alternative splicing of basic chitinase GENE PR3bin the low-nicotine mutants of Nicotiana tabacumL. cv. Burley 21
原文摘要：
Two unlinked semi-dominant loci, A (NIC1) and B (NIC2), control nicotine and related alkaloid biosynthesis in Burley tobaccos. Mutations in either or both loci (nic1 and nic2) lead to low nicotine phenotypes with altered environmental stress responses. Here we show that the transcripts derived from the pathogenesis-related (PR) protein gene PR3b are alternatively spliced to a greater extent in the nic1 and nic2 mutants of Burley 21 tobacco and the nic1nic2 double mutant. The alternative splicing results in a deletion of 65 nucleotides and introduces a premature stop codon into the coding region of PR3b that leads to a significant reduction of PR3b specific chitinase activity. Assays of PR3b splicing in F2 individuals derived from crosses between nic1 and nic2 mutants and wild-type plants showed that the splicing phenotype is controlled by the NIC1 and NIC2loci, even though NIC1 and NIC2 are unlinked loci. Moreover, the transcriptional analyses showed that the splicing patterns of PR3b in the low-nicotine mutants were differentially regulated by jasmonate (JA) and ethylene (ET). These data suggest that the NIC1 and NIC2 loci display differential roles in regulating the alternative splicing of PR3b in Burley 21. The findings in this study have provided valuable information for extending our understanding of the broader effects of the low-nicotine mutants of Burley 21 and the mechanism by which JA and ET signalling pathways post-transcriptionally regulate the activity of PR3b protein.
作者：张洪博
