| 毛竹肉桂酰辅酶A还原酶基因PeCCR功能初步研究 |
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| 引用本文： | 徐浩,杨克彬,朱成磊,李英,高志民.毛竹肉桂酰辅酶A还原酶基因PeCCR功能初步研究[J].林业科学研究,2020,33(2):77-84. |
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| 作者姓名： | 徐浩 杨克彬 朱成磊 李英 高志民 |
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| 作者单位： | 国际竹藤中心,竹藤资源基因科学与基因产业化研究所,国家林业和草原局/北京市竹藤科学与技术重点开放实验室,北京100102 |
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| 基金项目： | 国际竹藤中心基本科研业务费专项资金项目(1632019008)。 |
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| 摘 要： | 目的]为研究肉桂酰辅酶A还原酶(CCR)基因表达对竹子木质素生物合成的影响,对毛竹(Phyllostachys edulis (Carrière) J. Houz.)中PeCCR基因的表达情况进行分析,并对PeCCR基因功能进行研究,以期为利用CCR基因在竹子中开展基因工程育种提供参考依据。方法]采用实时定量PCR(qRT-PCR)方法对PeCCR基因在毛竹不同组织以及不同高度笋中的表达进行了分析,采用RT-PCR方法克隆了PeCCR基因的编码区,构建了基因过量表达载体,采用蘸花法转化拟南芥(Arabidopsis thaliana L.),采用溴乙酰法测定转基因植株茎木质素的含量。结果]qRT-PCR结果表明:在毛竹实生苗根中PeCCR的表达量最高,其次是笋中,而未展开叶中最低;在野外随着笋高度的增加,木质化程度加强,PeCCR基因的表达量呈上升趋势,在6.7 m笋中达到最高。克隆获得PeCCR编码区长度为1 026 bp,编码一个341 aa的蛋白,具有家族蛋白特有的保守结构域"NWYCYGK"。与野生型拟南芥相比,转PeCCR基因植株叶片明显增大,且抽苔时间提前3~4 d。茎横切的组织化学染色观察发现:转基因植株茎的木质部和束间纤维组织染色面积均大于野生型;木质素含量测定表明,2个过表达PeCCR1转基因株系中的木质素含量均明显高于野生型,分别为野生型对照的123.1%和116.7%。结论]PeCCR基因在毛竹不同组织的表达存在差异,在笋中随高度增加其表达量上调。过量表达PeCCR促进了转基因拟南芥植株的生长发育,提高了木质素含量。
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| 关 键 词： | 毛竹 肉桂酰辅酶A还原酶基因 表达分析 木质素 |
Preliminary Study on the Function of Cinnamoyl-CoA Reductase Gene PeCCR of Moso Bamboo(Phyllostachys edulis) |
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| XU Hao,YANG Ke-bin,ZHU Cheng-lei,LI Ying,GAO Zhi-min.Preliminary Study on the Function of Cinnamoyl-CoA Reductase Gene PeCCR of Moso Bamboo(Phyllostachys edulis)[J].Forest Research,2020,33(2):77-84. |
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| Authors: | XU Hao YANG Ke-bin ZHU Cheng-lei LI Ying GAO Zhi-min |
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| Affiliation: | (Institute of Gene Science and Industrialization for Bamboo and Rattan Resources,International Center for Bamboo and Rattan,State Forestry and Grassland Administration/Beijing Key Open Laboratory on the Science and Technology of Bamboo and Rattan,Beijing 100102,China) |
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| Abstract: | Objective] To reveal the effect of cinnamoyl-CoA reductase(CCR) gene expression on bamboo lignin,the expression of PeCCR in moso bamboo(Phyllostachys edulis) was analyzed, and the gene function of PeCCR was studied, which provided a reference for bamboo breeding using genetic engineering with CCR gene. Method] Realtime quantitative PCR(qRT-PCR) was used to analyze the expression of PeCCR in different tissues and the shoots with different heights of moso bamboo. The coding region of PeCCR was cloned by RT-PCR and its overexpression vector was constructed. PeCCR was transformed into Arabidopsis thaliana by floral dip method, and the content of lignin in transgenic plants was determined by bromoacetyl method. Result] The results of qRT-PCR showed that the expression level of PeCCR was the highest in the roots of moso bamboo seedling, followed by the shoots, and the lowest in the unexpanded leaves. Under the natural environment, with the increase of bamboo shoot height, the degree of lignification increased, and the gene expression of PeCCR showed an upward trend, reaching the highest in6.7 m bamboo shoots. The coding region of PeCCR was 1 026 bp, encoding a 341 aa protein with a conserved domain of "KNWYCYGK" unique to the CCR family. Phenotypic analysis showed that compared with the wild type,the leaves of PeCCR transgenic Arabidopsis plants became larger obviously, and the bolting time was 3-4 days earlier. The histochemical staining of stem transverse sections showed that the staining area of xylem and inter-fiber tissue in transgenic plants were larger than that of wild type. The lignin measurement demonstrated that the lignin content of two transgenic lines overexpressing PeCCR1 was significantly higher than that of wild type(123.1% and116.7% of the wild type control, respectively). Conclusion] PeCCR was differently expressed in different tissues of moso bamboo, and it was upregulated in bamboo shoots with increasing height. Overexpression of PeCCR promoted the growth and development of transgenic Arabidopsis plants with increased lignin content. |
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| Keywords: | Phyllostachys edulis Cinnamoyl-CoA reductase gene Expression analysis Lignin |
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