小麦类胡萝卜素生物合成关键酶基因的克隆与遗传转化研究
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摘要
小麦是世界上重要的粮食作物，提高其营养品质是小麦遗传育种研究的重要课题。类胡萝卜素是维生素A的前体，维生素A的缺乏可导致夜盲症、白内障等眼部疾病，甚至于完全失明，还会加剧腹泻、呼吸疾病、儿童麻疹等，但普通小麦中类胡萝卜素含量相对较低(3μg／g以下)。本文以小麦类胡萝卜素生物合成关键酶(八氢番茄红素合成酶PSY、八氢番茄红素脱氢酶PDS和ζ-胡萝卜素脱氢酶ZDS)的EST序列为切入点，采用分子生物学方法分离基因的cDNA全长序列，检测分析基因的表达特性，在此基础上利用转基因方法鉴定基因的功能，并探讨提高小麦类胡萝卜素含量的基因工程新途径；获得以下主要结论：
(1)采用RACE技术，首次从普通小麦中分离获得类胡萝卜素合成途径中两个重要脱氢酶基因TaPDS和TaZDS的cDNA全序列；同时，采用同源序列分离法，获得了小麦PSY基因的完整编码区序列。通过RT-PCR半定量法检测分析了PSY、PDS和ZDS基因的表达特性，发现三者在富含叶绿体、质体的光合作用器官中高水平表达，而在非光合作用的种子中表达较弱。首次证实了普通小麦种子中存在类胡萝卜素的代谢途径，且种子中类胡萝卜素含量较低，为提高种子类胡萝卜素含量的基因工程改良研究提供了理论基础。
(2)应用“最小基因表达框”的小麦转基因技术，利用小麦胚乳特异性强启动子和广谱性启动子分别驱动玉米的八氢番茄红素合成酶PSY1基因、欧文氏菌胡萝卜素脱氢酶CrtI基因在小麦中超量表达，获得转基因植株，转基因种子中类胡萝卜素含量约由0.5μg／g提高到3.8～4.9μg／g；首次鉴定了玉米PSY1基因和细菌CrtI基因在小麦中的功能，并且表明应用转基因技术提高类胡萝卜素含量的可行性。
(3)完善了“基因最小表达框”基因枪法小麦转基因技术体系，转化率达到0.7％，与常规转基因技术的转化率相一致，为进一步建立安全性转基因技术体系奠定了良好基础。
本文在类胡萝卜素代谢途径关键酶基因分离和功能分析的基础上，采用转基因技术提高了小麦种子类胡萝卜素含量，对于深入理解小麦类胡萝卜素合成的分子生物学基础和提高小麦营养品质的育种实践都具有一定意义。
Wheat is the word's important food crop, and improving the nutritional quality of wheat is an important objective in wheat molecular breeding programs. Carotenoids are essential components of human diets, primarily as precursors of vitamin A. Vitamin A deficiency causes symptoms ranging from night blindness to those of xerophthamia and keratomalacia, leading to total blindness. Furthermore, vitamin A deficiency exacerbates affilictiona, such as diarrhea, respiratory diseases, and childhood diseases such as measles. However, there is low carotenoid content in common wheat (less than 3μg/g).
In this thesis, firstly, the EST (Expressed Sequence Tag) sequences of several wheat carotenoids biosynthesis key enzymes, such as phytoene synthase (PSY), phytoene desaturase (PDS) andζ-carotene desaturase (ZDS), were analyzed. Then the full-length cDNA sequences and the expression characteristics of these genes were identified by molecular biology methods, on the basis of which, the function of these genes were identified in genetic transformation and the new pathway of increasing the wheat carotenoids content are discussed. The main results were as follows.
1. The full-length cDNA sequences of phytoene desaturase gene (TaPDS) andζ-carotene desaturase gene (TaZDS) were isolated from common wheat (Triticum aestivum. L.) by using RACE (Rapid Amplification of cDNA Ends) method. Otherwise, the complete coding sequence of phytoene synthase gene (PSY) was isolated from the same wheat variety by using homologous sequence separation. Through semi-quantitative RT-PCR method, the expressions of PSY, PDS and ZDS genes were detected. These genes showed higher expression in photosynthetic tissues (leaves and flowers), and lower expression in non-photosynthetic tissues (seeds). Since the first full-length cDNA cloning of these enzymes in common wheat are reported here, the implications for the genetic modification of the carotenoids (pro-vitamin A) content in wheat are also discussed.
2. In order to increase the carotenoid content of common wheat endosperm, transgenic wheat has been produced by over-expressing a maize psy1 gene coding phytoene synthase driven by endosperm-specific 1Dx5 promoter in the elite wheat (Triticum aestivum L.) variety EM12, together with a bacterial carotene dusaturase gene CrtI from Erinia uredovora under the constitutive CaMV 35S promoter control. This combination covers the requirements forβ-carotene synthesis and, as hoped, a clear increasing of carotenoids content was detected in the T_0 generation of transgenic wheat seeds, which showed a light yellow colour. We observed an increase in total carotenoids content from about 0.5μg/g up to 3.8～4.9μg/g.
3. The "minimal expression cassettes" transformation procedure has been developed and optimized. Stable transgenic plants were recovered from the immature embryos with transgenes on the linear gene cassettes lacking vector backbone sequences, corresponding to promoters, open reading frames and terminators. The efficiency of transformation using gene cassettes reached 0.7%, and it's similar to the conventional transgenic technology.
The results of present research provided with valuable reference for improving wheat nutritional quality through genetic engineering.
(1)采用RACE技术，首次从普通小麦中分离获得类胡萝卜素合成途径中两个重要脱氢酶基因TaPDS和TaZDS的cDNA全序列；同时，采用同源序列分离法，获得了小麦PSY基因的完整编码区序列。通过RT-PCR半定量法检测分析了PSY、PDS和ZDS基因的表达特性，发现三者在富含叶绿体、质体的光合作用器官中高水平表达，而在非光合作用的种子中表达较弱。首次证实了普通小麦种子中存在类胡萝卜素的代谢途径，且种子中类胡萝卜素含量较低，为提高种子类胡萝卜素含量的基因工程改良研究提供了理论基础。
(2)应用“最小基因表达框”的小麦转基因技术，利用小麦胚乳特异性强启动子和广谱性启动子分别驱动玉米的八氢番茄红素合成酶PSY1基因、欧文氏菌胡萝卜素脱氢酶CrtI基因在小麦中超量表达，获得转基因植株，转基因种子中类胡萝卜素含量约由0.5μg／g提高到3.8～4.9μg／g；首次鉴定了玉米PSY1基因和细菌CrtI基因在小麦中的功能，并且表明应用转基因技术提高类胡萝卜素含量的可行性。
(3)完善了“基因最小表达框”基因枪法小麦转基因技术体系，转化率达到0.7％，与常规转基因技术的转化率相一致，为进一步建立安全性转基因技术体系奠定了良好基础。
本文在类胡萝卜素代谢途径关键酶基因分离和功能分析的基础上，采用转基因技术提高了小麦种子类胡萝卜素含量，对于深入理解小麦类胡萝卜素合成的分子生物学基础和提高小麦营养品质的育种实践都具有一定意义。
Wheat is the word's important food crop, and improving the nutritional quality of wheat is an important objective in wheat molecular breeding programs. Carotenoids are essential components of human diets, primarily as precursors of vitamin A. Vitamin A deficiency causes symptoms ranging from night blindness to those of xerophthamia and keratomalacia, leading to total blindness. Furthermore, vitamin A deficiency exacerbates affilictiona, such as diarrhea, respiratory diseases, and childhood diseases such as measles. However, there is low carotenoid content in common wheat (less than 3μg/g).
In this thesis, firstly, the EST (Expressed Sequence Tag) sequences of several wheat carotenoids biosynthesis key enzymes, such as phytoene synthase (PSY), phytoene desaturase (PDS) andζ-carotene desaturase (ZDS), were analyzed. Then the full-length cDNA sequences and the expression characteristics of these genes were identified by molecular biology methods, on the basis of which, the function of these genes were identified in genetic transformation and the new pathway of increasing the wheat carotenoids content are discussed. The main results were as follows.
1. The full-length cDNA sequences of phytoene desaturase gene (TaPDS) andζ-carotene desaturase gene (TaZDS) were isolated from common wheat (Triticum aestivum. L.) by using RACE (Rapid Amplification of cDNA Ends) method. Otherwise, the complete coding sequence of phytoene synthase gene (PSY) was isolated from the same wheat variety by using homologous sequence separation. Through semi-quantitative RT-PCR method, the expressions of PSY, PDS and ZDS genes were detected. These genes showed higher expression in photosynthetic tissues (leaves and flowers), and lower expression in non-photosynthetic tissues (seeds). Since the first full-length cDNA cloning of these enzymes in common wheat are reported here, the implications for the genetic modification of the carotenoids (pro-vitamin A) content in wheat are also discussed.
2. In order to increase the carotenoid content of common wheat endosperm, transgenic wheat has been produced by over-expressing a maize psy1 gene coding phytoene synthase driven by endosperm-specific 1Dx5 promoter in the elite wheat (Triticum aestivum L.) variety EM12, together with a bacterial carotene dusaturase gene CrtI from Erinia uredovora under the constitutive CaMV 35S promoter control. This combination covers the requirements forβ-carotene synthesis and, as hoped, a clear increasing of carotenoids content was detected in the T_0 generation of transgenic wheat seeds, which showed a light yellow colour. We observed an increase in total carotenoids content from about 0.5μg/g up to 3.8～4.9μg/g.
3. The "minimal expression cassettes" transformation procedure has been developed and optimized. Stable transgenic plants were recovered from the immature embryos with transgenes on the linear gene cassettes lacking vector backbone sequences, corresponding to promoters, open reading frames and terminators. The efficiency of transformation using gene cassettes reached 0.7%, and it's similar to the conventional transgenic technology.
The results of present research provided with valuable reference for improving wheat nutritional quality through genetic engineering.
引文
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