【作者】 许明；

【导师】 郑金贵；

【作者基本信息】 福建农林大学 ， 作物遗传育种， 2011， 博士

【摘要】 水稻(Oryza sativa L.)是世界上最重要的粮食作物之一,稻米营养品质的优劣对人类健康和水稻生产具有重要的影响,其中蛋白质、必需氨基酸和微量元素是人们主要关注的稻米营养特性。稻米蛋白质中的赖氨酸、苏氨酸、蛋氨酸和色氨酸等人体必需氨基酸含量偏低,以致其蛋白质利用率低,营养不够完全。所以,提高稻米蛋白质中的必需氨基酸含量,显得十分必要。AmA1是一种分离于籽粒苋(Amaranthus L.)种子的人体必需氨基酸平衡蛋白质,该蛋白中的8种人体必需氨基酸含量均超过FAO/WHO推荐的理想蛋白质标准,且对人体不会造成过敏,在改良作物种子蛋白质营养上具有重要的应用前景。人乳铁蛋白(human lactoferrin,hLF)具有多种生物学功能,不仅可以提高铁营养,还具有广谱抗病菌、调节机体免疫反应等功能,该生物学功能若能为水稻所利用,将大大增加稻米内铁的生物有效性。受到种质资源及种间生殖隔离等因素的限制,利用常规育种手段改善稻米的营养品质,存在一定困难。本研究通过转基因技术将必需氨基酸平衡蛋白基因AmA1和人乳铁蛋白基因hLF转入水稻,使其在水稻种子中特异性高效表达,以达到改进稻米必需氨基酸组成和提高铁营养的目的。主要研究结果如下:1、为了使异源蛋白基因AmA1和hLF在转基因水稻种子中特异性高效表达并积累。本研究首先根据已报道的水稻种子特异性启动子核苷酸序列,从水稻中克隆了2个谷蛋白基因(GluB-1、Gt1)启动子和1个油膜蛋白基因Ole18启动子序列,并将这3个启动子以及组成型启动子CaMV 35S,分别与报告基因GUS相连再导入同一水稻品种中,以确定这些启动子的表达强弱及特异性。序列分析和转基因水稻的GUS活性分析结果表明:(1)从水稻“日本晴”克隆中的Gt1和ole18启动子与已报道序列完全一致,从水稻“台粳9号”中克隆的GluB-1启动子序列与已报道序列存在部分碱基差异,二者同源性为97%。启动子预测软件NNPP、顺式组件分析以及转基因水稻实验证实,该启动子能引导GUS基因在水稻胚乳中特异性表达,碱基差异没有对其功能造成影响。(2)Gt1和GluB-1基因启动子调控GUS基因在水稻胚乳中表达的活性强弱差异不大,但都明显高于CaMV35S启动子;Gt1启动子和Ole18启动子都具有严格的组织表达特异性,其中Gt1启动子引导GUS基因仅在水稻胚乳中特异性表达,Ole18启动子则能引导GUS基因仅在水稻胚和种皮中表达。2、根据已知的籽粒苋AmA1基因序列,从籽粒苋“千穗谷NO1”种子中克隆了AmA1基因的开放阅读框序列。序列分析表明,所克隆的AmA1基因的开放阅读框序列与已报道序列基本一致,仅存在1个碱基差异,但未改变氨基酸的编码。应用分子伴侣载体pBB540、pBB542和原核表达载体pET28a(+)进行了AmA1基因的原核表达及融合蛋白纯化。结果表明,经过IPTG的诱导,AmA1基因在宿主大肠杆菌中能正确表达出与预期大小一致(37kD)的融合蛋白,借助分子伴侣载体可以显著提高AmA1融合蛋白的可溶性。3、对已报道的植物基因组DNA快速提取方法作了进一步简化和改进,建立了一种适于大规模转基因水稻PCR检测的微量DNA提取法。通过DNA提取质量和PCR扩增效果比较发现,用该方法提取的水稻基因组DNA完整性较好,PCR扩增效果优于碱裂解法,与试剂盒提取法无明显的差异,结果稳定可靠;而且整个提取过程操作简单,花费时间少,平均一个样品只需5~6min;起始植物材料用量少,仅需5~10mg。4、构建了Gt1和Ole18基因启动子分别驱动AmA1基因表达的双T-DNA、双MARs植物表达载体PCDMAR-pGt1-AmA1-hpt和PCDMAR-pOle18-AmA1-hpt,并采用农杆菌介导法,将它们转入水稻明恢86和台粳9号中。PCR和Southern blot检测表明,以上两种载体携带的AmA1基因均已整合到水稻基因组中。Westhern blot检测结果显示,Gt1和Ole18启动子都能引导AmA1基因在水稻种子中表达。T1代转基因稻米的氨基酸含量检测结果表明:(1)在Gt1启动子调控AmA1基因表达的转基因水稻中,有9个株系种子中的7种必需氨基酸含量(占种子总干重)比对照有不同程度地提高,最大提高幅度为44.44%;有2个株系种子总氨基酸中的三种限制性必需氨基酸含量(即占总氨基酸的百分比)均比对照显著提高,其中赖氨酸最大提高了5.58%,苏氨酸最大提高了3.12%,甲硫氨酸最大提高了5.58%。(2)在Ole18启动子调控AmA1基因表达的转基因水稻中,有5个转基因株系种子中的7种必需氨基酸含量比对照有不同程度提高,最大提高幅度为15.56%,但只有1个株系的赖氨酸占总氨基酸的百分比对照提高了4.4%,其它株系种子总氨基酸中的必需氨基酸含量没有明显变化。以上结果说明,在Gt1启动子的调控下,AmA1基因能在水稻胚乳中特异性表达,且对提高水稻种子总氨基酸中的3种限制性必需氨基酸含量,具有一定的效果。5、在不改变氨基酸序列的前提下,根据水稻密码子的偏好性对人乳铁蛋白基因hLF和籽粒苋AmA1基因序列进行了优化设计,调整并去除了会影响基因转录、翻译效率及mRNA稳定性的序列元件,同时对这两个基因的5′端和3′端进行了适当修饰:(1)hLF基因经优化后,编码区核苷酸序列与原来一致性为76.3%,692个密码子中有431个被改变,总的G+C含量和密码子第三位G+C含量分别由原来的53.82%、59.88%提高到56.28%、66.38%;原基因中存在的3个PPSS序列和3处AT富集区全部被去除。并将其5′端的信号肽替换成谷蛋白基因GluB-1的信号肽序列,在3′端添加了一段内质网滞留信号KDEL序列和两个终止密码子TGATAA。(2)AmA1基因优化后、编码区核苷酸序列与原来保持了76.0%的一致性,305个密码子中有192个被改变,总的G+C含量和第三位G+C含量分别从原来的34.43%、29.51%提高到49.95%、71.48%;原基因中存在的17个PPSS序列、3处ATTTA序列和2处AT富集区分别减少到优化后的5、0和1;并在5′端添加了谷蛋白基因Gt1的信号肽序列。6、人工合成了优化后的hLF、AmA1基因(命名为omhLF、omAmA1)。为了比较hLF、AmA1基因优化前后在水稻中的表达水平差异,分别将优化前后的hLF、AmA1基因转入水稻台粳9号中,共获得PCR阳性植株178株。同时构建了含有omhLF、omAmA1双基因的双T-DNA植物表达载体,并通过农杆菌介导法转入水稻蜀恢527中,经PCR检测,有34株水稻转化植株能扩增出与预期大小一致的omhLF、omAmA1基因片段。由于上述转基因水稻植株目前正处在T0代分蘖期,对其进一步的分子鉴定和品质分析需要过一段时间才能进行。更多还原

【Abstract】 Rice (Oryza sativa L.) is one of the most important food crops. The nutritional quality of rice has great influence on human health and rice production. The protein, essential amino acid, microelement are the nutritional qualities of rice that are maily concernted.The contents of essential amino acids such as lysine, serine, methione and tryptophan, etc. are relatively low in rice grain, which decreases the utilization effiecency of protein and the nutritional value. Thus, it is necessary to increase the contents of essential amino acid content of rice grain so that the nutritional value can be improved. The AmA1 is a protein isolated from the seed of Amaranthus L. that posesses blanced essential amino acids, the contents of all eight essential amino acids are above the ideal protein standard that is recommended by the FAO/WHO. Also, this protein is friendly for human digestion and absorption. These properties have made the AmA1 a promising protein in the improvement of the nutritional quality of the crop seed protein. Anther problem of the nutritional quality of the rice grain is that the iron content is relatively low as well. The human lactoferrin (hLF) has a number of biochemical functions that are capable of not only improving iron nutrition but also bearing broad spectrum of antibacteria and adjusting the immune response of human body, etc. The application of hLF to rice will increase the biological effectiveness of iron in human body.The improvement of rice nutritional quality with conventional breeding methods has made quite a lot of progress. However, due to the inadequacy of rice germplasms and reproductive isolation, the application of conventional breeding methods has only limited success. The development of genetic engineering technique provides a feasible method for improving the nutritional quality of rice grain. In an attempt to improve the compositon of essential amino acids of rice grain and increase iron nutrition, the AmA1 and hLF gene were transformed to rice with transgenic technique in order to express the proteins in rice seed with high efficiency and specificity in this study. The main results are as follows:1.In order to lead AmA1 and hLF expressed and accumulated in rice seed with high efficiency and specificity. the two promoters of rice glutelin gene(GluB-1 and Gt1) and one promoter of oil membrane gene ole18 were cloned in this study according to the nucleotide sequences of the promoter of rice glutelins that are publicly available. The above three pomoters and the constitutive promoter CaMV 35S were ligated with reporter gene GUS and transformed to rice respecitively to determine the expression intensity and specifictiy. The results of sequence analysis and GUS assay of transgenic rice showed that: (1) The promoter sequenceses of Gt1 and ole18 cloned from rice cultivar“Nipponbare”were identical with the reported sequences. The promoter sequence of the GluB-1 cloned from rice cultivar “TG9”has a homology of 97% with the reported sequence. The analysis of promoter prediction software NNPP and cis-acting element and transgenic rice experiment indictated that the promote could guide the GUS gene to express specifically in rice embryo and the nucleotide sequence difference did not affect its function in this study. (2) The expression intensities of the GUS gene with regulation of Gt1 and GluB-1 did not show much difference, but they were both higher than that of the CaMV35S. The Gt1 and Ole18 both have stringent tissue specificity of expression. The Gt1 guided the specific expression of GUS in rice embryo and the Ole 18 guided the specific expression of GUS in rice embryo and seed capsule.2.The OFR sequence of the AmA1 was cloned from the seed of Amaranthus L. cultivar“Amaranthus hypochondriacus L.NO1”. The sequence analysis showed that the cloned sequence isessentially identical with the reported sequence. There was only one nucleotide difference but the amino acid coding was the same. The molecular chaperonevectors pBB540 and pBB542 were applied to assist the soluble expression of the pET28a-AmA1 in E. coli and the target protein was purified with Ni-NTA affinitychromatography. The results indicated that the IPTG could induce the production of the recombinant protein,of which the moleclar weight was consistent with the the oretical molecular weight of 37kDa. Thus, the utilization of the molecular chaperone vector could significantly improve the the soluble expression of the recombinant protein AmA1.3.The reported method of wheat rapid DNA extraction was simplified at some steps and then applied to rice in this study. The results showed that rice genomic DNA extracted by this simplified method was intergal and the PCR amplification using it as template was stable and reliable, showing no significant difference from plant genomic DNA extraction kit method. Furthermore, the whole extraction procedure is simple, takes less time(only 5~6 min per sample on average), and requires a small amount of plant sample about 5~10mg. Therefore this method is suitable for the large-scale PCR detection of transgenic rice.4.The plant expression vectors with two T-DNA and dual MARs were constructed, which were PCDMAR-pGt1-AmA1-hpt and and PCDMAR-pOle18-AmA1-hpt under the control of Gt1 promoter and Ole18 promoter respecitively, and transformed to MH86 and TG9 with agrobacterium mediated method. The PCR andSouthern blot analysis showed that the AmA1 genes in the two vectors were integrated to the rice genome. The Western blot results showed that the Gt1 promoter and Ole18 promoter could guide the correct expression of AmA1 in rice seed. The assay of the amino acid content in the grain of T1 transgenic rice showed: (1) seven amino acids contents (accounting for the total dry weight) in the 9 lines in which the AmA1 expression was regulated with Gt1 promoter were higher than those of the control to various degrees, the largest of which was 44.44%. The contents (accounting for the percentage in the total amino acids) of three essential amino acids (lysin,threonine and methionine) in the lines were significantly higher than those of the control, with the maximun increase of lysine 5.58%, threonine 3.12% and methionine5.58%. (2) seven amino acids contents (accounting for the total dry weight) in the 5 lines in which the AmA1 gene expression was regulated with Ole18 promoter were higher than those of the control to various degrees, the largest of which was 15.56% and there was only one strain that hasa 4.4% increase of lysine content in the total amino acids. The above results suggested that the AmA1 gene could express specifically in rice embryo under the regulation of Gt1 promoter and the contents of the three essential amino acids were increased in a notable extent. 5.Under the premise of not altering the amino acid sequence, codon usage of native hLF and AmA1 genes are optimizied according to rice preferred codons. The sequence elements that affect the efficiency of gene transcription, translation and stability of mRNA are readjusted or removed purposefully for efficient plant expression. The 5′and 3′ends of the gene are modified at the same time. The main results are listed as follows:(1) The optimized and modified hLF gene shares 76.3% nucleotide sequence similarity to the wild hLF gene in the coding region. Of the 692 codons for the mature peptide of hLF gene, 431 codons are changed. The ration of total G+C content and G+C cotent of bases at the third position in codons are increased from 53.82%, 59.88% of the wile type to 56.28% ,66.38% of the modified. Three of PPSS (potential polyadneylylation signal sequence) and 3 of A+T rich regions are removed entirely. Then for the optimized outer-gene code, we strip the rice GluB-1 gene signal peptide sequences in the 5′end,KDEL and double terminators sequences in the 3’the end,respectively. (2) The optimized and modified AmA1 gene share 76.0% nucleotide sequence similarity to the wild AmA1 gene in the coding region. Of the 305 codons for the mature peptide of AmA1 gene, 192 codons are changed. The ration of total G+C content and G+C cotent of bases at the third position in codons are increased from 34.43% and 29.51% of the wild type to 49.95%, 71.48% of the modified. The number of PPSS,ATTTA sequences and A+T rich regions are decreased from 17,3 and 2 to 5, 0 and 1, respectively. Then for the optimized outer-gene code, we strip the rice Gt1 gene signal peptide sequences in the 5’end.6. The optimized hLF ( named omhLF) and AmA1 ( named omAmA1) gene are synthesized. Then the native and optimized hLF,AmA1 gene are transferred into rice cultivar“TG9”respectively for the purpose of comparing their expression level difference in rice. 178 PCR-positive line are obtained. Meanwhile, we construct a plant expression vector containing omhLF and omAmA1 gene and transfers it into rice cultivar“SH527”by agrobacterium-mediated method. The result of PCR detection shows that fragment of omhLF and omAmA1 gene with expected size are amplified from 34 transgenic lines. Due to be at tillering stage of T0 generation, further molecular identification and quality analysis for these lines will be conducted in the next time.更多还原