特异启动子控制的GhGA20ox1基因对棉花纤维发育的影响
摘要
棉花是世界上最重要的天然纤维作物,也是最重要的经济作物。我国是世界上最大的纺织品生产国和消费国,棉花产业在我国的国民经济中具有举足轻重的地位。近年来,随着人民生活水平的提高和纺织技术的革新,对棉花产量和品质提出了更高的要求。一个优良的棉花品种,不仅要产量高,而且纤维品质要好,方能实现其最终产品价值。基因工程育种技术可以打破物种间的遗传障碍,实现优良目的基因的定向转移,同时具有后代易于稳定,育种周期短等优点,这为棉花纤维产量和品质的改良提供了新的途径。
赤霉素(Gibberellins,GAs)是一类重要的植物激素,影响着高等植物生活史的各个阶段,如种子萌发,茎的伸长,叶的伸展,花器官的诱导和发育及种子和果实的形成。研究表明,棉花纤维细胞的起始与伸长与IAA、GAs和BR等植物激素相关。在培养基中添加GA和IAA,培养的胚珠着生纤维的数量显著增加;在田间自然生长状态下,对开花前或开花后的花蕾和棉铃外施GA和IAA,可以明显增加纤维细胞的数量。本实验室前期克隆了棉花GA20-氧化酶基因(GhGA20ox1),并发现超量表达该基因能促进棉花GA合成,进而促进纤维起始和早期伸长。本论文在前期研究的基础上,深入分析了GhGA20ox1基因的超量表达和RNA干扰对棉花(特别是纤维)发育的影响,进一步研究了Fbp7、E6和Sub启动子驱动的GhGA20ox1正义与反义基因对棉花生长发育的影响。主要结果如下:
观察发现,GhGA20ox1超量表达的棉花表现出GA过量表达的性状,如:植株变高、节间伸长、叶片变小、现蕾开花晚、结实率低,并且纤维较野生型略长,种子略小。
通过遗传转化,共获得特异启动子Fbp7、E6和Sub调控下正义和反义的GhGA20ox1转基因棉花植株83株,GUS检验均为阳性。经RT-PCR分析,找出了基因表达量相对提高和降低的株系。对基因表达量相对提高和降低的株系进行Southern blotting分析,证实了GhGA20ox1基因均以单拷贝的形式整合到棉花基因组中。对转基因植株表型的观察,未发现特异启动子Fbp7、E6和Sub调控下的GhGA20ox1正义和反义表达的植株与野生型植株有显著差异。
对Fbp7:GA20和Fbp7:AntiGA20植株0DPA的胚珠进行扫描电镜观察,发现Fbp7:AntiGA20的转基因棉花胚珠表面纤维细胞突起数目减少,35S:GA20的纤维突起明显。对35S:GA20、35S:GA20RNAi、Fbp7:GA20和Fbp7:AntiGA20植株1DPA的胚珠进行石蜡切片观察,发现35S:GA20RNAi的纤维突起数目较少,其余转基因棉花株系未观察到明显差异。对35S:GA20、35S:GA20RNAi、Fbp7:GA20、Fbp7:AntiGA20、E6:AntiGA20、Sub:GA20和Sub:AntiGA20转基因棉花成熟纤维数目进行统计,发现Fbp7:AntiGA20和35S:GA20RNAi转基因棉花植株单粒种子着生的长纤维数量都比对照少。
对转基因棉花纤维进行品质检测,结果表明,Fbp7:GA20转基因棉花纤维的细度和强度均比对照有所改良,但改良程度不及超量表达植株。
上述结果说明,GhGA20ox1基因在棉花纤维发育和品质形成中具有重要的作用。
Cotton fiber is an important raw material for textile industry, and cotton has an important strategic status in our national economy. In recent years, textile industry focus their economic considerations on fiber quality, and consistently demand better lint quality. Because of the negative relationship between fiber productivity and quality; cotton breeding nowadays faces a big challenge to simultaneously improve cotton yield and fiber quality. Genetic engineering provided a new strategy and excellent perspectives to cotton breeders. These limitations in traditional breeding can be overcome through genetic engineering, which enables the transfer of genes from any source into cotton and changes the negative associability of yield and quality.
Gibberellins are endogenous hormones controlling numerous aspects of plant growth and development such as seeds germination, stem elongation, leaf shape and the development of flower and fruit. Pre- and post-anthesis application of exogenous GA can improve fiber production in upland cotton. A cotton GA 20-oxidase gene (GhGA20ox1) was cloned previously in our lab, and overexpression of this gene promote GA biosynthesis in cotton, and further fiber initiation and elongation at the early stage. On the basis of previous researches, the phenotypic variations in the GhGA20ox1 overexpressing was analyzed in depth, and by cotton transformation, the influence of Fbp7-, E6-, and Sub-driven GhGA20ox1 on fiber development was further studied in the present thesis.
The main results are as following:
35S:GA20 transgenic cotton plants showed GA-overproduction phenotype. This phenotype includes higher plant, longer internodes and fiber, smaller leaf and seed, later flowering and lower boll setting rate.
A total of eighty-three GUS positive primary (T_0) transgenic lines (Fbp7:GA20, Fbp7:AntiGA20, E6:GA20, E6:AntiGA20, Sub:GA20 and Sub:AntiGA20 ) were generated in this study. RT-PCR was employed to assess GhGA20ox1 mRNA levels in these transgenic cottons, and several lines with enhanced or reduced expression of targeted genes were identified and were used to analyze the effects of transgenes on cotton development. Southern blotting analysis indicated that there was single copy of T-DNA present in the investigated cotton lines.
There were no significant phenotypic changes in plant growth observed between wild-type and Fbp7-, E6-, and Sub-driven GhGA20ox1 transformed cottons. Scanning electron microscope (SEM) observation indicated that the number of fibers initial decreased in 0-DPA ovules of Fbp7:AntiGA20 transgenic cotton, but fiber initiation showed no obvious changes in Fbp7:GA20 cottons, compared to the control ovules. Furthermore, the 1DPA ovules of 35S:GA20, 35S:GA20RNAi, Fbp7:GA20 and Fbp7:AntiGA20 cottons were observed by optical microscope. Only the fibers initiation in 35S:GA20RNAi ovules were found to be reduced.
The number of mature fiber from the 35S:GA20, 35S:GA20RNAi, Fbp7:GA20, Fbp7:AntiGA20, E6:AntiGA20, Sub:GA20 and Sub:AntiGA20 plants was further investigated. It was found that the number of mature fiber per seed in Fbp7:AntiGA20 and 35S:GA20RNAi transgenic plants were significantly decreased compared to the control.
Cotton fiber quality test displayed that the fineness and strength of Fbp7: GA20 fiber was slightly improved compared with control fiber, consistent with the improved quality of 35S: GA20 transgenic fiber.
Our results demonstrated that the GhGA20ox1 gene play an important role in fiber development and the quality formation in cotton.
赤霉素(Gibberellins,GAs)是一类重要的植物激素,影响着高等植物生活史的各个阶段,如种子萌发,茎的伸长,叶的伸展,花器官的诱导和发育及种子和果实的形成。研究表明,棉花纤维细胞的起始与伸长与IAA、GAs和BR等植物激素相关。在培养基中添加GA和IAA,培养的胚珠着生纤维的数量显著增加;在田间自然生长状态下,对开花前或开花后的花蕾和棉铃外施GA和IAA,可以明显增加纤维细胞的数量。本实验室前期克隆了棉花GA20-氧化酶基因(GhGA20ox1),并发现超量表达该基因能促进棉花GA合成,进而促进纤维起始和早期伸长。本论文在前期研究的基础上,深入分析了GhGA20ox1基因的超量表达和RNA干扰对棉花(特别是纤维)发育的影响,进一步研究了Fbp7、E6和Sub启动子驱动的GhGA20ox1正义与反义基因对棉花生长发育的影响。主要结果如下:
观察发现,GhGA20ox1超量表达的棉花表现出GA过量表达的性状,如:植株变高、节间伸长、叶片变小、现蕾开花晚、结实率低,并且纤维较野生型略长,种子略小。
通过遗传转化,共获得特异启动子Fbp7、E6和Sub调控下正义和反义的GhGA20ox1转基因棉花植株83株,GUS检验均为阳性。经RT-PCR分析,找出了基因表达量相对提高和降低的株系。对基因表达量相对提高和降低的株系进行Southern blotting分析,证实了GhGA20ox1基因均以单拷贝的形式整合到棉花基因组中。对转基因植株表型的观察,未发现特异启动子Fbp7、E6和Sub调控下的GhGA20ox1正义和反义表达的植株与野生型植株有显著差异。
对Fbp7:GA20和Fbp7:AntiGA20植株0DPA的胚珠进行扫描电镜观察,发现Fbp7:AntiGA20的转基因棉花胚珠表面纤维细胞突起数目减少,35S:GA20的纤维突起明显。对35S:GA20、35S:GA20RNAi、Fbp7:GA20和Fbp7:AntiGA20植株1DPA的胚珠进行石蜡切片观察,发现35S:GA20RNAi的纤维突起数目较少,其余转基因棉花株系未观察到明显差异。对35S:GA20、35S:GA20RNAi、Fbp7:GA20、Fbp7:AntiGA20、E6:AntiGA20、Sub:GA20和Sub:AntiGA20转基因棉花成熟纤维数目进行统计,发现Fbp7:AntiGA20和35S:GA20RNAi转基因棉花植株单粒种子着生的长纤维数量都比对照少。
对转基因棉花纤维进行品质检测,结果表明,Fbp7:GA20转基因棉花纤维的细度和强度均比对照有所改良,但改良程度不及超量表达植株。
上述结果说明,GhGA20ox1基因在棉花纤维发育和品质形成中具有重要的作用。
Cotton fiber is an important raw material for textile industry, and cotton has an important strategic status in our national economy. In recent years, textile industry focus their economic considerations on fiber quality, and consistently demand better lint quality. Because of the negative relationship between fiber productivity and quality; cotton breeding nowadays faces a big challenge to simultaneously improve cotton yield and fiber quality. Genetic engineering provided a new strategy and excellent perspectives to cotton breeders. These limitations in traditional breeding can be overcome through genetic engineering, which enables the transfer of genes from any source into cotton and changes the negative associability of yield and quality.
Gibberellins are endogenous hormones controlling numerous aspects of plant growth and development such as seeds germination, stem elongation, leaf shape and the development of flower and fruit. Pre- and post-anthesis application of exogenous GA can improve fiber production in upland cotton. A cotton GA 20-oxidase gene (GhGA20ox1) was cloned previously in our lab, and overexpression of this gene promote GA biosynthesis in cotton, and further fiber initiation and elongation at the early stage. On the basis of previous researches, the phenotypic variations in the GhGA20ox1 overexpressing was analyzed in depth, and by cotton transformation, the influence of Fbp7-, E6-, and Sub-driven GhGA20ox1 on fiber development was further studied in the present thesis.
The main results are as following:
35S:GA20 transgenic cotton plants showed GA-overproduction phenotype. This phenotype includes higher plant, longer internodes and fiber, smaller leaf and seed, later flowering and lower boll setting rate.
A total of eighty-three GUS positive primary (T_0) transgenic lines (Fbp7:GA20, Fbp7:AntiGA20, E6:GA20, E6:AntiGA20, Sub:GA20 and Sub:AntiGA20 ) were generated in this study. RT-PCR was employed to assess GhGA20ox1 mRNA levels in these transgenic cottons, and several lines with enhanced or reduced expression of targeted genes were identified and were used to analyze the effects of transgenes on cotton development. Southern blotting analysis indicated that there was single copy of T-DNA present in the investigated cotton lines.
There were no significant phenotypic changes in plant growth observed between wild-type and Fbp7-, E6-, and Sub-driven GhGA20ox1 transformed cottons. Scanning electron microscope (SEM) observation indicated that the number of fibers initial decreased in 0-DPA ovules of Fbp7:AntiGA20 transgenic cotton, but fiber initiation showed no obvious changes in Fbp7:GA20 cottons, compared to the control ovules. Furthermore, the 1DPA ovules of 35S:GA20, 35S:GA20RNAi, Fbp7:GA20 and Fbp7:AntiGA20 cottons were observed by optical microscope. Only the fibers initiation in 35S:GA20RNAi ovules were found to be reduced.
The number of mature fiber from the 35S:GA20, 35S:GA20RNAi, Fbp7:GA20, Fbp7:AntiGA20, E6:AntiGA20, Sub:GA20 and Sub:AntiGA20 plants was further investigated. It was found that the number of mature fiber per seed in Fbp7:AntiGA20 and 35S:GA20RNAi transgenic plants were significantly decreased compared to the control.
Cotton fiber quality test displayed that the fineness and strength of Fbp7: GA20 fiber was slightly improved compared with control fiber, consistent with the improved quality of 35S: GA20 transgenic fiber.
Our results demonstrated that the GhGA20ox1 gene play an important role in fiber development and the quality formation in cotton.
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