中文
隨著植物抗病基因工程的發展,越來越多的抗病基因被人們所發現。2004年,Taler等從印度野生瓜的霜黴病抗性品種中克隆得到兩個具有絲氨酸乙醛酸轉氨酶(serineglyoxylate aminotransferase,SGT)活性的甜瓜霜黴病抗性基因At1和At2。Taler等發現At1和At2具有絲氨酸乙醛酸轉氨酶活性與植物光呼吸途徑相關,不屬於任何壹類已知R基因,對病原菌的抵抗沒有種屬專化性,並且它們的抗病作用與H_2O_2相關,基於此種現象,他們提出壹種新的抗病機制“酶抗病性”,這是首次報道的通過改變酶的表達量而賦予植物抗病性。據推測At1和At2還可能對霜黴病以外的其它多種植物葉部病害具有抗性作用,是很有研究價值的抗病基因,並且,“酶抗病性”作用機制的提出還需要更多的基因證據和試驗支持。據此本論文從光呼吸作用非常強的大豆中克隆At1和At2的同源基因GmSGT,對其進行序列分析、酶活性中心預測、原核表達分析,並將其轉入受體植物中進行抗病性鑒定。現將本論文的主要研究結果及創新點總結如下: 1.利用大豆EST序列和5'-Race技術,首次從大豆霜黴病抗性品種中克隆了編碼絲氨酸乙醛酸轉氨酶的GmGGT1基因序列(已獲得國家發明專利1項,專利號:ZL2005100887 83.4),同時從受SA誘導的大豆霜黴病感病品種黑農10號中克隆得到了兩條GmSGT1的同源基因GmSGT2,GmSGT3。序列分析表明,GmSGT1與Taler等報道的甜瓜霜黴病抗病基因At1、At2氨基酸序列同源性達88.03%和87.78%;同時,GmSGT1與擬南芥(Arabidopsis thaliana L.)、水稻(Oryzae sativa L.)、貝母(Fritillariaagrestis)、紫萍(Spirodela polyrrhiza)的絲氨酸乙醛酸轉氨酶同源性達83.33%-85.79%。GmSGT1推導蛋白序列分析顯示它具有壹個5磷酸吡哆醛結合位點GSQKAL和壹個強的過氧化物體定位信號SRI,表明該基因可能在植物的過氧化物體中通過光呼吸途徑起作用。GmSGT2和GmSGT3與GmSGT1核苷酸序列同源性高達96.38%和99.17%,推導的氨基酸序列同源性為97.03%和99.25%。利用生物信息學方法對GmSGT1,GmSGT2,GmSGT3蛋白的酶學活性中心進行了分析,結果顯示三個蛋白序列均具有絲氨酸乙醛酸轉氨酶活性。 2.首次通過存大腸桿菌中模擬植物光呼吸途徑的技術,證實了GmSGT1具有絲氨酸乙醛酸轉氨酶活性。光呼吸途徑中發生的反應:乙醇酸(?)H_2O_2+乙醛酸;乙醛酸(?)甘氦酸。因大腸桿菌中含有乙醇酸氧化酶(GOX),所以向原核表達菌株中加入乙醇酸,可完成上述反應,通過檢測H_2O_2量的變化,可確定GmSGT1基因的絲氨酸乙醛酸轉氨酶功能。本研究結果顯示,只有表達GmSGT1蛋白的組分能誘導大量H_2O_2產生,對照組分無,因此,可以確定GmSGT1具有絲氨酸乙醛酸轉氨酶活性。 3.分析了GmSGT蛋白與大豆不同品種的抗性相關性。Western雜交結果顯示,抗霜黴病品種早豐5號和九農9號中可檢測到有目的蛋白的表達,而感病品種黑農10號中無表達。SA誘導前、後半定量RT-PCR結果表明,感病品種黑農10號在SA誘導前未檢測到表達,而SA誘導後有微量表達,大豆對霜酶病的抗病性也有大幅度提高。因此,我們推斷,GmSGH的表達確實和SA誘導途徑相關,並且隨著GmSGT1表達水平的提高,大豆對霜黴病的抗病性也明顯提高。 4.構建了GmSGT1植物表達載體,進行煙草轉化,獲得了轉基因植株,並對其進行煙草赤星病,黑脛病,青枯病的抗性鑒定,結果表明轉基因煙草顯著提高了煙草對赤星病,黑脛病,青枯病的抗性。本研究為Taler等提出的植物“酶抗性基因”提供了新的實驗證據。 5.利用抗病品種早豐5號探索了GmSGT1基因在大豆中的時空表達特性,該基因在大豆葉片中有表達,而根,莖中無表達,並且隨著生育期的增強而表達增強,生殖生長期最強,然後隨著細胞的老化而表達減弱,直至消失。這表明GmSGT1的變化趨勢與植物光呼吸的變化趨勢相符。同時檢測了光呼吸途徑中在絲氨酸乙醛酸轉氨酶上遊起作用的乙醇酸氧化酶(GOX)的表達特性,結果表明GOX的表達水平與GmSGT1表達水平的變化具有壹致性,說明GmSGT1表達水平提高的同時,它的上遊反應也增強,H_2O_2表達也提高。這與我們推測的GmSGT蛋白在植物光呼吸途徑中起作用的結論相符。 6.研究確定了大豆遺傳轉化體系。建立了大豆早豐5號,黑農10號的胚芽尖組培體系,並利用農桿菌LBA4404介導法將構件好的植物表達載體pIM1.1-GmSGT-plus轉化早豐5號,RNAi植物表達載體pIM1.1-GmSGT-plusF轉化黑農10號,獲得了再生植株。同時,對早豐5號的胚芽尖,子葉節,胚軸三種外植體在再生頻率,再生時間,K篩選濃度,農桿菌不同侵染時間對重生芽再生頻率的影響進行了研究,為早豐5號組培,轉化體系的進壹步優化提供了實驗依據。
英文
With the development of plant disease resistance genes, more and more resistance genes are found in people. In 2004, Taler and other wild melon from India, downy mildew resistant varieties have cloned two serine glyoxylate aminotransferase (serineglyoxylate aminotransferase, SGT) activity Melon downy mildew resistance genes At1 and At2. Taler At1 and At2 is found with serine glyoxylate aminotransferase activity and plant photorespiration pathway, does not belong to any class of known R genes, resistance to the pathogen resistance without special species, and their role in disease resistance associated with H_2O_2 Based on this phenomenon, they proposed a new resistance mechanism "enzyme resistance", this is the first reported expression of the enzyme by changing the plant disease resistance conferred.It is speculated that At1 and At2 may also downy mildew than many other leaf diseases of plants resistant to the effect that the great research value of the resistance genes, and the "resistance enzyme" mechanism of the proposed need for more many genes to support evidence and testing. Pursuant to which the paper is very strong from the soybean photorespiration clone At1 and At2 in the homologous gene GmSGT, its sequence analysis, enzyme active site predicted prokaryotic expression analysis, and transferred to the receptor for disease resistance in plants identification.Now the paper's major findings and innovation are summarized as follows: 1. Using soybean EST sequences, and 5'-Race technology for the first time from downy mildew resistant varieties of soybean clone encoding serine glyoxylate aminotransferase gene sequences GmGGT1 (has received a national invention patent, patent number: ZL2005100887 83.4),Induced by SA from both downy mildew susceptible varieties of soybean, 10 black farmers have been two GmSGT1 cloned the homologous gene GmSGT2, GmSGT3. Sequence analysis revealed that, GmSGT1 and Taler other melon downy mildew resistance genes reported At1, At2 amino acid sequence homology of up to 88.03% and 87.78%; the same time, GmSGT1 and Arabidopsis (Arabidopsis thaliana L.), rice (Oryzae sativa L.), Fritillaria (Fritillariaagrestis), Spirodela (Spirodela polyrrhiza) serine glyoxylate aminotransferase homology 83.33% -85.79%.GmSGT1 deduced protein sequence analysis revealed that it has a 5-pyridoxal phosphate binding sites GSQKAL and a strong peroxisome targeting signal SRI, showed that the gene may be in the plant peroxisome photorespiration way to work through. GmSGT2 and GmSGT3 and GmSGT1 nucleotide sequence homology as high as 96.38% and 99.17%, the deduced amino acid sequence homology was 97.03% and 99.25%. By bioinformatics method GmSGT1, GmSGT2, GmSGT3 protein enzyme active sites were analyzed, the results show that the three protein sequences all have serine glyoxylate aminotransferase activity.2. For the first time through the simulation of plant survival in E. coli pathway of light breathing techniques, confirmed the GmSGT1 with serine glyoxylate aminotransferase activity. Photorespiratory pathway reaction occurs: glycolic acid (?) H_2O_2 + glyoxylic acid; glyoxylate (?) Gan helium acid. Because E. coli contains glycolic acid oxidase (GOX), so the original strain by adding acid expression, to be completed by the above-mentioned reaction, by detecting changes in the amount H_2O_2 can determine GmSGT1 serine glyoxylate aminotransferase gene function. The results showed that only the expression of GmSGT1 protein component can induce a large number of H_2O_2 production, control components not, therefore, can determine GmSGT1 glyoxylate aminotransferase activity with serine.3. Analysis of the GmSGT protein soybean varieties with resistance to relevance. Western blotting results showed that downy mildew resistant cultivars EARLY 5 and 9 farmers could be detected in 9 purposeful expression, while black farmers on the 10th susceptible and no expression. SA induction before and after the semi-quantitative RT-PCR results showed that the susceptible black farmers on the 10th in SA was not detected before the induction of expression, while SA induced the expression of a trace of soybean on the cream of the resistance enzymes have greatly improved . Therefore, we conclude, GmSGH SA induced the expression of true and relevant way, and with the expression level of GmSGT1 increased soybean downy mildew resistance was also improved significantly.4. Constructed GmSGT1 plant expression vector for tobacco transformation, we obtained the transgenic plants, and gain tobacco brown spot, black shank, bacterial wilt resistance identification, the results showed that the transgenic tobacco significantly increased the tobacco brown spot, black shank, bacterial wilt resistance. Taler, etc. This study proposed plant "enzyme resistance genes," provides a new experimental evidence. 5. Use of resistant varieties as early as 5 to explore the GmSGT1 Feng gene expression in the temporal and spatial characteristics of soybean, the gene expression in soybean leaves in, but the roots, stems and no expression, and increased with the growth period while the expression, most reproductive stage, and then with the decreased expression of cell aging, until the disappearance.This shows that the trend of plant GmSGT1 Photorespiration trend line. Simultaneous detection of the photorespiratory pathway of serine glyoxylate aminotransferase in the upper reaches of the role of glycolate oxidase (GOX) of the expression characteristics, the results show that the expression level of GOX GmSGT1 consistent changes in expression levels, indicating enhanced expression GmSGT1 At the same time, it also increased the upstream response, H_2O_2 expression also increased. We speculate that the GmSGT This plant photorespiratory pathway proteins play a role in the conclusion.6. Study identified the genetic transformation system. HSBC was established as early as 5, soybean, black farmers on the 10th of the embryonic tip tissue culture system, using Agrobacterium tumefaciens LBA4404 mediated plant expression vector will be a good member pIM1.1-GmSGT-plus conversion EARLY 5, RNAi plant expression vector pIM1.1-GmSGT-plusF transformed black farmers on the 10th, won the regeneration. Meanwhile, HSBC on the 5th of the embryo as early as sharp, cotyledonary node, hypocotyl explants of three in the regeneration frequency, regeneration time, K screening concentration, Agrobacterium infection time on the regeneration of different frequencies of shoot regeneration was studied for EARLY 5 tissue culture, transformation system provides an experimental basis for further optimization.