race技术论文参考文献

这就是：中文 随着植物抗病基因工程的发展,越来越多的抗病基因被人们所发现。2004年,Taler等从印度野生瓜的霜霉病抗性品种中克隆得到两个具有丝氨酸乙醛酸转氨酶(serineglyoxylate aminotransferase,SGT)活性的甜瓜霜霉病抗性基因At1和At2。Taler等发现At1和At2具有丝氨酸乙醛酸转氨酶活性与植物光呼吸途径相关,不属于任何一类已知R基因,对病原菌的抵抗没有种属专化性,并且它们的抗病作用与H_2O_2相关,基于此种现象,他们提出一种新的抗病机制“酶抗病性”,这是首次报道的通过改变酶的表达量而赋予植物抗病性。据推测At1和At2还可能对霜霉病以外的其它多种植物叶部病害具有抗性作用,是很有研究价值的抗病基因,并且,“酶抗病性”作用机制的提出还需要更多的基因证据和试验支持。据此本论文从光呼吸作用非常强的大豆中克隆At1和At2的同源基因GmSGT,对其进行序列分析、酶活性中心预测、原核表达分析,并将其转入受体植物中进行抗病性鉴定。现将本论文的主要研究结果及创新点总结如下: 1.利用大豆EST序列和5'-Race技术,首次从大豆霜霉病抗性品种中克隆了编码丝氨酸乙醛酸转氨酶的GmGGT1基因序列(已获得国家发明专利1项,专利号:ZL2005100887 83.4),同时从受SA诱导的大豆霜霉病感病品种黑农10号中克隆得到了两条GmSGT1的同源基因GmSGT2,GmSGT3。序列分析表明,GmSGT1与Taler等报道的甜瓜霜霉病抗病基因At1、At2氨基酸序列同源性达88.03%和87.78%;同时,GmSGT1与拟南芥(Arabidopsis thaliana L.)、水稻(Oryzae sativa L.)、贝母(Fritillariaagrestis)、紫萍(Spirodela polyrrhiza)的丝氨酸乙醛酸转氨酶同源性达83.33%-85.79%。GmSGT1推导蛋白序列分析显示它具有一个5磷酸吡哆醛结合位点GSQKAL和一个强的过氧化物体定位信号SRI,表明该基因可能在植物的过氧化物体中通过光呼吸途径起作用。GmSGT2和GmSGT3与GmSGT1核苷酸序列同源性高达96.38%和99.17%,推导的氨基酸序列同源性为97.03%和99.25%。利用生物信息学方法对GmSGT1,GmSGT2,GmSGT3蛋白的酶学活性中心进行了分析,结果显示三个蛋白序列均具有丝氨酸乙醛酸转氨酶活性。 2.首次通过存大肠杆菌中模拟植物光呼吸途径的技术,证实了GmSGT1具有丝氨酸乙醛酸转氨酶活性。光呼吸途径中发生的反应:乙醇酸(?)H_2O_2+乙醛酸;乙醛酸(?)甘氦酸。因大肠杆菌中含有乙醇酸氧化酶(GOX),所以向原核表达菌株中加入乙醇酸,可完成上述反应,通过检测H_2O_2量的变化,可确定GmSGT1基因的丝氨酸乙醛酸转氨酶功能。本研究结果显示,只有表达GmSGT1蛋白的组分能诱导大量H_2O_2产生,对照组分无,因此,可以确定GmSGT1具有丝氨酸乙醛酸转氨酶活性。 3.分析了GmSGT蛋白与大豆不同品种的抗性相关性。Western杂交结果显示,抗霜霉病品种早丰5号和九农9号中可检测到有目的蛋白的表达,而感病品种黑农10号中无表达。SA诱导前、后半定量RT-PCR结果表明,感病品种黑农10号在SA诱导前未检测到表达,而SA诱导后有微量表达,大豆对霜酶病的抗病性也有大幅度提高。因此,我们推断,GmSGH的表达确实和SA诱导途径相关,并且随着GmSGT1表达水平的提高,大豆对霜霉病的抗病性也明显提高。 4.构建了GmSGT1植物表达载体,进行烟草转化,获得了转基因植株,并对其进行烟草赤星病,黑胫病,青枯病的抗性鉴定,结果表明转基因烟草显著提高了烟草对赤星病,黑胫病,青枯病的抗性。本研究为Taler等提出的植物“酶抗性基因”提供了新的实验证据。 5.利用抗病品种早丰5号探索了GmSGT1基因在大豆中的时空表达特性,该基因在大豆叶片中有表达,而根,茎中无表达,并且随着生育期的增强而表达增强,生殖生长期最强,然后随着细胞的老化而表达减弱,直至消失。这表明GmSGT1的变化趋势与植物光呼吸的变化趋势相符。同时检测了光呼吸途径中在丝氨酸乙醛酸转氨酶上游起作用的乙醇酸氧化酶(GOX)的表达特性,结果表明GOX的表达水平与GmSGT1表达水平的变化具有一致性,说明GmSGT1表达水平提高的同时,它的上游反应也增强,H_2O_2表达也提高。这与我们推测的GmSGT蛋白在植物光呼吸途径中起作用的结论相符。 6.研究确定了大豆遗传转化体系。建立了大豆早丰5号,黑农10号的胚芽尖组培体系,并利用农杆菌LBA4404介导法将构件好的植物表达载体pIM1.1-GmSGT-plus转化早丰5号,RNAi植物表达载体pIM1.1-GmSGT-plusF转化黑农10号,获得了再生植株。同时,对早丰5号的胚芽尖,子叶节,胚轴三种外植体在再生频率,再生时间,K筛选浓度,农杆菌不同侵染时间对重生芽再生频率的影响进行了研究,为早丰5号组培,转化体系的进一步优化提供了实验依据。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.

这也行？？？早知道当初我也百度提问了。费了我一周时间自己翻译5000字段英文文献。

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