白菜春化相关基因BcFLC的克隆与功能分析
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摘要
芸薹属(Brassica)蔬菜是我国栽培面积最大、总产量最高的一类蔬菜作物。其中白菜(Brassicacampestris L.ssp.chinensis Makino,syn.B.rapa ssp.chinensis)类蔬菜绝大部分属于冬性一年生蔬菜作物,需要经历一个低温阶段(春化作用)才能顺利完成阶段发育转变。感温先期抽薹是影响普通白菜(Brassica campestris ssp.chinensis vat.communis)产量的一个重要因素,也是生产上迫切需要解决的问题。因而研究普通白菜的开花习性进而培育晚抽薹品种是解决这一问题的根本途径。菜心(B.campestris ssp.chinensis var.parachinensis)是白菜的一个变种,它对低温要求不严格,在华南地区可以周年生产。是代表极端早抽薹的—类白菜类作物。普通白菜和菜心具有相似的遗传背景,对春化需求却表现出巨大的差异,这为我们在白菜类蔬菜中研究春化作用提供了良好的材料。随着分子生物学理论和技术的发展,对同为十字花科的模式植物拟南芥(Arabidopsis thaliana)春化作用的研究已经十分深入,这也为我们的研究提供了便利。本实验室在前一阶段实验中利用同源基因克隆法和RACE技术,从普通白菜和菜心中克隆到了拟南芥春化核心基因FLC的同源基因BcFLC-1和BcFLC-2,以及它们的内含子1序列。为进一步了解BcFLC基因的功能以及普通白菜与菜心对春化需求不同的原因,我们进而克隆了BcFLC的启动子序列,并构建了BcFLC基因的正义和反义载体,通过农杆菌介导的方法分别转入菜心和普通白菜中,得到了230株转化植株。分别对它们的开花时间进行观察。取得结果如下:
(1)应用生物信息学软件对BcFLC-1与BcFLC-2基因编码区的核苷酸序列进行分析,发现两者编码序列的一致性高达99.34%,氨基酸序列包含与开花密切相关的MADS-盒结构域和K-盒结构域,仅在保守区域外存在1个氨基酸的差异。两者的内含子1序列的相似性达91.67%,启动子序列的相似性达97.1%,表明BcFLC-1和BcFLC-2并不存在实质性的差异。
(2)分别构建了正义BcFLC基因以及反义BcFLC基因的组成型启动子CaMV35S的表达载体pBI35S-BcFLC1和pBI35S-BcFLC2。检测鉴定后分别将它们导入农杆菌LBA4404菌株中。
(3)根据余小林等(2002)构建的菜心和普通白菜遗传转化体系,共获得48个芽系的230株抗卡那霉索抗性苗。其中转正义载体的菜心20个芽系,转化效率为3.608%,转空载体对照的菜心2个芽系.转化效率为1.831%,转反义载体的普通白菜18个芽系,转化效率为2.272%,转空载体对照的普通白菜2个芽系,转化效率为1.210%。以芽系为单位提取DNA,经PCR检测、Southern印迹杂交,所得到的再生植株中84.78%为阳性转基因植株。
(4)观察转基因菜心和普通白菜的开花时间发现,转入正义BcFLC基因表达载体的菜心平均在分化后54 d、长出10片真叶时现蕾,而对照转空载体的菜心平均在分化后的第45 d、8片真叶时现蕾。转入反义BcFLC基因表达载体的普通白菜与对照转空载体的普通白菜的开花时间差异明显,转基因普通白菜在无需春化的情况下,平均在分化后78 d、10片真叶时现蕾,继而抽薹开花,蕾期自交授粉后可正常结实。而对照植株不经春化作用,一直保持营养生长状态。
Plants in Brassica genus are a kind of vegetable crops with the highest yield and are widely cultivated throughout China. One of the major kinds of Brassica is Chinese cabbage-pak-choi (Brassica campestris L. ssp. chinensis Makino, syn. B. rapa ssp. chinensis), most of which own a winter annual flowering habit. It is a habit that plants needs a period of cold (vernalization) to transfer from vegetative growth to reproductive growth, and to complete the life cycle. In practice, premature bolting is a serious problem not only causes a great of loss in production, but also reduces the commercial value. Anti-bolting breeding is the ultimate approach to solve this problem, which calls for the understanding of these species' flowering habits. Flowering Chinese cabbage (B. campestris ssp. chinensis var. parachinensis) is a unique variety, which does not need vernalization to induce flowering, the so called summer annual flowering habit. It represents of an extreme early-bolting species, which can be produced all over the year in southern parts of China. Common Chinese cabbage-pak-choi (Brassica campestris ssp. chinensis var. communis) has a similar genetic background with flowering Chinese cabbage, but it greatly differs from the latter in the requirement of vernalization. They provide us excellent materials in the study of vernalization in Brassica crops. Along with the development of molecular biological theories and technologies, the molecular mechanism of vernalization in Arabidopsis thaliana, which also belongs to Cruciferae, has been studied in-depth by many researchers. We could use the above resources for reference in our studies.
By homologous cloning and rapid amplification of cDNA ends (RACE), vernalization-related gene BcFLC-1 and BcFLC-2 and their intron 1 sequences were already isolated from B. campestris ssp. chinensis var. communis cv. Shanghai-qing and B. campestris ssp. chinensis var. parachinensis cv. Sijiu, respectively. In order to fully understand the function of BcFLC and the causation of different requirement of vernalization between common Chinese cabbage-pak-choi and flowering Chinese cabbage, we cloned the promoter of BcFLC, and constructed plant expressing plasmid vectors of anti-sense RNA and over-expression vectors using fragments of BcFLC. By infecting common Chinese cabbage-pak-choi and flowering Chinese cabbage through Agrobacterium-medmted transformation, respectively. the transgenic plants of common Chinese cabbage-pak-choi and flowering Chinese cabbage were obtained consequently. And the flowering habits of the transgenic plants were observed subsequently. The results provided available information on exploring the function of BcFLC and the mechanism of vernalization in Brassica crops. The results are as follows:
(1) The nucleotide sequences of the coding areas of BcFLC-1 and BcFLC-2 were analyzed by bioinformatics software, the results of which showed a conformity of 99.34%. MADS-box and K-box, which were closely related to flowering in Arabidopsis, existed in the same location of the both amino acids sequences. Only one amino acid difference was found outside the conservative regions. The two intron 1 sequences showed a similarity of 91.67%, while the two promoter sequences got a 97.1% consistency All these indicated that BcFLC-1 and BcFLC-2 had no substantial difference.
(2) The over-expression and anti-sense RNA expressing plasmid vectors with CaMV 35S promoter, pBI35S-BcFLC1 and pBI35S-BcFLC2 were constructed using fragments of BcFLC. Molecular identification showed that both of the vectors along with the pBI35S (negative control) were introduced into Agrobacterium tumefaciens strain LBA4404.
(3) 230 Kan~R plantlets of 48 regenerated lines were obtained on the efficient genetic transformation system based on Yu XL et al (2002). There were 20 regenerated lines of pBI35S-BcFLC1 plantlets with an efficiency of 3.608% and 2 regenerated lines of pB1121 plantlets with an efficiency of 1.831% in flowering Chinese cabbage. Similar situation happened in common Chinese cabbage-pak, while there were 18 regenerated lines of pB135S-BcFLC2 plantlets with an efficiency of 2.272% and 2 regenerated linos of pB1121 plantlets with an efficiency of 1.210%. The frequency of positive Kan~R seedlings is 84 78% examined by PCR and Southern blot.
(4) Flower buds in transgenic flowering Chinese cabbage showed 54 days after differentiating culture or when the 10~(th) leaf came out, while the control showed flower buds 45 days after differentiating culture or when the 8~(th) leaf came out. Transgenic common Chinese cabbage-pak-choi displayed great difference with the control, by showing flower buds without vernalization on an average of 78 days after differentiating culture of when the 10~(th) leaf came out.
(1)应用生物信息学软件对BcFLC-1与BcFLC-2基因编码区的核苷酸序列进行分析,发现两者编码序列的一致性高达99.34%,氨基酸序列包含与开花密切相关的MADS-盒结构域和K-盒结构域,仅在保守区域外存在1个氨基酸的差异。两者的内含子1序列的相似性达91.67%,启动子序列的相似性达97.1%,表明BcFLC-1和BcFLC-2并不存在实质性的差异。
(2)分别构建了正义BcFLC基因以及反义BcFLC基因的组成型启动子CaMV35S的表达载体pBI35S-BcFLC1和pBI35S-BcFLC2。检测鉴定后分别将它们导入农杆菌LBA4404菌株中。
(3)根据余小林等(2002)构建的菜心和普通白菜遗传转化体系,共获得48个芽系的230株抗卡那霉索抗性苗。其中转正义载体的菜心20个芽系,转化效率为3.608%,转空载体对照的菜心2个芽系.转化效率为1.831%,转反义载体的普通白菜18个芽系,转化效率为2.272%,转空载体对照的普通白菜2个芽系,转化效率为1.210%。以芽系为单位提取DNA,经PCR检测、Southern印迹杂交,所得到的再生植株中84.78%为阳性转基因植株。
(4)观察转基因菜心和普通白菜的开花时间发现,转入正义BcFLC基因表达载体的菜心平均在分化后54 d、长出10片真叶时现蕾,而对照转空载体的菜心平均在分化后的第45 d、8片真叶时现蕾。转入反义BcFLC基因表达载体的普通白菜与对照转空载体的普通白菜的开花时间差异明显,转基因普通白菜在无需春化的情况下,平均在分化后78 d、10片真叶时现蕾,继而抽薹开花,蕾期自交授粉后可正常结实。而对照植株不经春化作用,一直保持营养生长状态。
Plants in Brassica genus are a kind of vegetable crops with the highest yield and are widely cultivated throughout China. One of the major kinds of Brassica is Chinese cabbage-pak-choi (Brassica campestris L. ssp. chinensis Makino, syn. B. rapa ssp. chinensis), most of which own a winter annual flowering habit. It is a habit that plants needs a period of cold (vernalization) to transfer from vegetative growth to reproductive growth, and to complete the life cycle. In practice, premature bolting is a serious problem not only causes a great of loss in production, but also reduces the commercial value. Anti-bolting breeding is the ultimate approach to solve this problem, which calls for the understanding of these species' flowering habits. Flowering Chinese cabbage (B. campestris ssp. chinensis var. parachinensis) is a unique variety, which does not need vernalization to induce flowering, the so called summer annual flowering habit. It represents of an extreme early-bolting species, which can be produced all over the year in southern parts of China. Common Chinese cabbage-pak-choi (Brassica campestris ssp. chinensis var. communis) has a similar genetic background with flowering Chinese cabbage, but it greatly differs from the latter in the requirement of vernalization. They provide us excellent materials in the study of vernalization in Brassica crops. Along with the development of molecular biological theories and technologies, the molecular mechanism of vernalization in Arabidopsis thaliana, which also belongs to Cruciferae, has been studied in-depth by many researchers. We could use the above resources for reference in our studies.
By homologous cloning and rapid amplification of cDNA ends (RACE), vernalization-related gene BcFLC-1 and BcFLC-2 and their intron 1 sequences were already isolated from B. campestris ssp. chinensis var. communis cv. Shanghai-qing and B. campestris ssp. chinensis var. parachinensis cv. Sijiu, respectively. In order to fully understand the function of BcFLC and the causation of different requirement of vernalization between common Chinese cabbage-pak-choi and flowering Chinese cabbage, we cloned the promoter of BcFLC, and constructed plant expressing plasmid vectors of anti-sense RNA and over-expression vectors using fragments of BcFLC. By infecting common Chinese cabbage-pak-choi and flowering Chinese cabbage through Agrobacterium-medmted transformation, respectively. the transgenic plants of common Chinese cabbage-pak-choi and flowering Chinese cabbage were obtained consequently. And the flowering habits of the transgenic plants were observed subsequently. The results provided available information on exploring the function of BcFLC and the mechanism of vernalization in Brassica crops. The results are as follows:
(1) The nucleotide sequences of the coding areas of BcFLC-1 and BcFLC-2 were analyzed by bioinformatics software, the results of which showed a conformity of 99.34%. MADS-box and K-box, which were closely related to flowering in Arabidopsis, existed in the same location of the both amino acids sequences. Only one amino acid difference was found outside the conservative regions. The two intron 1 sequences showed a similarity of 91.67%, while the two promoter sequences got a 97.1% consistency All these indicated that BcFLC-1 and BcFLC-2 had no substantial difference.
(2) The over-expression and anti-sense RNA expressing plasmid vectors with CaMV 35S promoter, pBI35S-BcFLC1 and pBI35S-BcFLC2 were constructed using fragments of BcFLC. Molecular identification showed that both of the vectors along with the pBI35S (negative control) were introduced into Agrobacterium tumefaciens strain LBA4404.
(3) 230 Kan~R plantlets of 48 regenerated lines were obtained on the efficient genetic transformation system based on Yu XL et al (2002). There were 20 regenerated lines of pBI35S-BcFLC1 plantlets with an efficiency of 3.608% and 2 regenerated lines of pB1121 plantlets with an efficiency of 1.831% in flowering Chinese cabbage. Similar situation happened in common Chinese cabbage-pak, while there were 18 regenerated lines of pB135S-BcFLC2 plantlets with an efficiency of 2.272% and 2 regenerated linos of pB1121 plantlets with an efficiency of 1.210%. The frequency of positive Kan~R seedlings is 84 78% examined by PCR and Southern blot.
(4) Flower buds in transgenic flowering Chinese cabbage showed 54 days after differentiating culture or when the 10~(th) leaf came out, while the control showed flower buds 45 days after differentiating culture or when the 8~(th) leaf came out. Transgenic common Chinese cabbage-pak-choi displayed great difference with the control, by showing flower buds without vernalization on an average of 78 days after differentiating culture of when the 10~(th) leaf came out.
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