苜蓿叶绿素合成酶(CHLG)编码基因的表达及对生物量的影响
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国家自然科学基金 (31772663);


Expression of Chlorophyll Synthase (CHLG) Encoding Gene and Its Effect on Biomass in Medicago
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    摘要:

    叶绿素由一系列酶促反应催化生成,其最后一步是叶绿素合成酶(chlorophyll synthase, CHLG)催化合成叶绿素a和叶绿素b。该实验主要以蒺藜苜蓿(Medicago truncatula)生态型R108和两种不同秋眠级紫花苜蓿(Medicago sativa)为材料,采用生物信息学方法对蒺藜苜蓿叶绿素合成酶(MtCHLG)的进化关系和基因结构进行分析,采用实时荧光定量方法分析MtCHLG对光照/黑暗、脱落酸(ABA)及聚乙二醇(PEG)的响应,利用叶绿素酸酯a加氧酶(MtCAO)突变体确定其与MtCHLG的上下游关系,并分析豆科牧草紫花苜蓿中MsCHLG表达量与其株高和产量的关系。结果表明:(1)植物CHLG功能域保守,其基因结构高度一致,植物在向陆生进化的过程中,CHLG基因数目略有增加,内含子长度明显增长。(2)克隆到MtCHLG的CDS为1,137 bp,其编码378个氨基酸,蛋白与紫花苜蓿MsCHLG相似性高达99.2%。(3)qRTPCR分析显示,MtCHLG基因主要在蒺藜苜蓿幼嫩叶中表达且受光诱导和黑暗抑制,表现为显著的昼增夜降的表达特征;100 μmol·L-1的ABA在短时间(2 h)处理后叶片中MtCHLG基因表达量下降为对照的26%(P<0.05);5%的PEG长时间(24 h)处理后MtCHLG基因表达量降低至对照的42%(P<0.05)。(4)瞬时表达重组蛋白MtCHLGGFP于烟草(Nicotiana benthamiana)表皮细胞,荧光检测结果显示,该蛋白主要定位在叶绿体上。(5)定量分析发现,mtcao突变体中几乎检测不到MtCAO基因的表达,且MtCHLG基因的表达水平约为野生型的44%,证明MtCAO功能缺失导致了MtCHLG转录水平降低,表明MtCHLG位于MtCAO的下游。(6)高秋眠级紫花苜蓿材料的株高为低秋眠级材料的2.5~3.5倍,其平均产量约为低秋眠级材料的2.1倍,且前者中MsCHLG基因的表达量略高于后者(1.2~1.5倍);相关性分析发现,紫花苜蓿中MsCHLG基因的表达水平与株高、产量成正相关。研究暗示,MsCHLG表达可作为苜蓿产量早期预测的参考指标,CHLG基因也可作为培育高产豆科饲草的备选基因。

    Abstract:

    Chlorophyll biosynthesis is catalyzed by a series of enzymes. Among them, the chlorophyll synthase (CHLG) catalyzes the synthesis of chlorophyll a and b, the last step of the pathway. In this study, Medicago truncatula (ecotype R108) and Medicago sativa with fall dormancy of 4 or 8 were used. The evolutionary relationship of plant CHLGs and their gene structure were analyzed using bioinformatics; the expression of MtCHLG under light/darkness, abscisic acid (ABA) or polyethylene glycol (PEG) treatment was studied by realtime quantitative PCR; the transcript level of MtCHLG was tested in mtcao mutant; and the correlation coefficient between the expression level of MsCHLG and plant height or yield in Medicago sativa was analyzed. The results showed that: (1) CHLGs shared common conserved domains and a relatively uniform gene composition. It seems that several terrestrial plants possess one more CHLG with much longer introns. (2) MtCHLG open reading frame (ORF) of 1 137 bp was cloned encoding 378 amino acids and the protein was highly identical (99.2%) to MsCHLG. (3) qRTPCR demonstrated that MtCHLG was predominantly expressed in young leaves of Medicago truncatula with a lightinducible or darkrepressed pattern showing a significant increase in the daytime and a continuous decline at night. Moreover, under 100 μmol·L-1 ABA treatment of 2 h, MtCHLG in leaves decreased to about 26% of the control (P<0.05). 5% PEG treatment within 24 h repressed the expression of MtCHLG, which decreased to about 42% of the control (P<0.05) at the end of the treatment. (4) When transiently expressed in tobacco epidermal cells, the recombinant protein MtCHLGGFP was detected mainly in chloroplasts. (5) qRTPCR showed that in mtcao mutant with no obvious expression of MtCAO, the expression level of MtCHLG was about 44% of the wild type, indicating that MtCAO lossoffunction resultes in a decrease of MtCHLG, which suggests that MtCHLG is downstream of MtCAO in the chlorophyll biosynthesis pathway. (6) The plant height of the alfalfa accessions with FD of 8 was about 2.5-3.5 times of the ones with FD of 4. The average yield of the former was about 2.1 times of the latter, and the expression level of MsCHLG showed a similar pattern (1.2-1.5 times). According to the correlation coefficient, the expression level of MsCHLG was significantly correlated with both plant height and yield. The findings suggest that MsCHLG transcriptional level might be useful for preliminary prediction of alfalfa yield at early stage, and CHLG may serve as a candidate gene to improve yield of legume forages by breeding.

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孔 杰,刘文文,蒋学乾,等.苜蓿叶绿素合成酶(CHLG)编码基因的表达及对生物量的影响[J].西北植物学报,2021,41(7):1079-1090

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  • 在线发布日期: 2021-08-19
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