转盐生杜氏藻β-胡萝卜素羟化酶基因(chyb)烟草的获得及耐盐性鉴定

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摘要 β-胡萝卜素羟化酶(β-carotene hydroxylase, chyb)作为盐生杜氏藻(Dunaliella viridis)胡萝卜素合成代谢途径中的关键酶，催化β-胡萝卜素经两步反应合成玉米黄素。盐生杜氏藻chyb基因在植物抗逆性方面有重要研究价值。本研究利用前期克隆得到的盐生杜氏藻chyb基因(GenBank登录号: JN118489)，构建了植物表达载体pCAMBIA3301-chyb。通过根癌农杆菌(Agrobacterium tumefaciens)LBA4404介导的叶盘法将其转入烟草(Nicotiana tabacum)，获得16株草铵膦抗性再生植株，经PCR及RT-PCR扩增分析表明，chyb基因已转入其中13株烟草植株中，并可以进行正常转录。通过测定转基因烟草的株高、叶面积、叶绿素含量、叶片相对电导率和脯氨酸含量对转基因烟草的耐盐性进行了检测，结果表明，与对照组相比，转基因烟草的株高、叶面积、叶绿素含量均极显著提高(P＜0.01)，其中转基因烟草平均株高为37.82 cm，是对照组烟草平均株高的1.37倍。在100 mmol/L NaCl胁迫处理下，转基因烟草的成活率为84.45%，是对照组烟草的1.58倍；在200 mmol/L NaCl胁迫下对照组烟草萎蔫死亡，而转基因烟草仍可生长存活。经检测转基因烟草叶片的相对电导率升高但变化幅度较小，脯氨酸含量较对照组极显著增加(P＜0.01)。研究结果说明转基因烟草的耐盐性得到了提高，为进一步探讨盐生杜氏藻chyb基因在植物耐盐性方面的功能提供基础资料。

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关键词 ： β-胡萝卜素羟化酶基因(chyb), 载体构建, 转化, 耐盐性

Abstract：β-carotene hydroxylase (chyb) catalyzes the conversion of β-carotene to zeaxanthin through β-cryptoxanthin in the pathway of carotenoid biosynthesis. The salt-tolerance of chyb gene in Dunaliella viridis has important research value in the aspect of plant resistance. chyb gene (GenBank accession No. JN118489) cloned in previous research was used to construct the plant expression vector pCAMBIA3301-chyb and transferred into tobacco (Nicotiana tabacum) by Agrobacterium tumefaciens LBA4404, and 16 regenerated tobaccos resisted to glufosinate ammonium were obtained. The results of PCR and RT-PCR indicated that chyb gene had been transferred successfully into 13 of them. Besides, salt tolerance of transgenic tobacco was detected through measuring plant height, leaf area, chlorophyll content, leaf relative conductivity and proline content. The result showed that plant height, leaf area and chlorophyll content were extremely significantly increased (P＜0.01) compared with the control group. The average height of transgenic plant was 37.82 cm, which was 1.37 times higher than that of the control group. Transgenic tobaccos were treated with different concentrations of NaCl. The results demonstrated that the average survival rate of transgenic tobacco was 1.58 times higher than that of control group under 100 mmol/L NaCl stress. Transgenic tobacco could grow under the treatment of 200 mmol/L NaCl, while the growth of control tobacco was inhibited. The leaf relative conductivity increased slightly, and the proline content of transgenic plants were improved extremly significantly than the control group (P＜0.01). It was concluded that chyb gene could improve salt-tolerance in transgenic tobacco plants and all the results help to lay the foundation for further exploring salt-tolerance of chyb gene.

Key words： β-carotene hydroxylase gene (chyb) Vector construction Transformation Salt tolerance

收稿日期: 2015-01-14 出版日期: 2015-07-09

基金资助:浙江省科技厅重点科技创新团队自主设计项目;宁波科技攻关项目

通讯作者: 龚一富 E-mail: gongyifu@163.com

引用本文:

许峰龚一富刘林周静孙娇璐王何瑜. 转盐生杜氏藻β-胡萝卜素羟化酶基因(chyb)烟草的获得及耐盐性鉴定 [J]. , 2015, 23(9): 1149-1156.

链接本文:

http://journal05.magtech.org.cn/Jwk_ny/CN/ 或 http://journal05.magtech.org.cn/Jwk_ny/CN/Y2015/V23/I9/1149

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