转Bt/CpTI基因水稻知识产权调查报告

水稻是世界上最重要的粮食作物之一.自1988年首次获得转基因水稻以来,水稻转基因技术已获得突飞猛进的发展,在抗虫、抗病、抗除草剂、抗逆和改良稻米品质转基因等研究方面皆有长足进步.转基因水稻研发中涉及许多专利问题.本文以转Bt基因水稻、转CpTI基因水稻和转Bt/CpTI双价基因水稻为研究对象,对研究中所涉及的知识产权问题进行了调查.结果表明,我国在转基因水稻研究中的大多数材料和技术含有国外机构所持有的专利.本文对产生的原因和可能存在的问题进行了讨论,并对解决方式提出了建议.     

最新转基因水稻、玉米、小麦成果简介

自1983年世界第一种转基因作物问世以来,全球转基因农作物种植已达到5000万公顷以上,获得新基因610个,其中包括水稻分蘖基因MOC1、融合抗虫基因CryCI、隐性抗水稻白叶枯病基因xa5和xa13等一批具有重要应用价值并拥有自主知识产权的新基因46个。     

转多个抗真菌蛋白基因水稻植株的获得及其抗稻瘟病菌的？…

用基因枪法将水稻碱性几丁质酶（ＲＣ２４）基因、芷蓿葡聚糖酶（β－Ｇｌｕ）基因、大麦核糖体失活蛋白（Ｂ－ＲＩＰ）基因和潮霉素基因同时导入籼稻品种（七丝软占）中,获得了个潮霉素抗性再生系,Ｓｏｕｔｈｅｒｎ ｂｌｏｔ证明２－３个抗真菌蛋白基因已整合到水稻基因组中,初步抗性鉴定表明ＲＯ代转基因水稻植株对稻瘟病菌的抗生有所提高。     

拟南芥NCED3基因在水稻中的过量表达可以提高水稻的干旱胁迫耐受性

9-顺-环氧类胡萝卜素双加氧酶(NCED)是高等植物脱落酸(ABA)生物合成途径中的关键酶,其催化的裂解反应直接生成ABA的前体物质.本文应用拟南芥rd29A诱导型启动子和35S组成型启动子成功地将AtNCED3基因在水稻中过量表达,通过耐旱性筛选实验证明转基因水稻对干旱胁迫的耐受性有了显著提高,并且该优良性状在T2代中得到稳定遗传.进一步的分析表明,过量表达AtNCED3可以促进转基因水稻种子的休眠;在含rd29A诱导型启动子的转基因水稻中检测到ABA下游基因OsBZ8的表达;推测 AtNCED3在水稻中的过量表达可能会提高水稻中内源 ABA的水平,从而提高植株的耐旱性.     

褐飞虱喂养试验显示表达GNA的转基因水稻纯系抗褐飞虱

利用基因枪法将含有3个不同基因（hpt,gus和gna）的质粒pWRG1515和pRSSGNA1共同转化粳稻品种鄂晚5号成熟胚诱导的愈伤组织。共再生出35株独立转基因植株。PCR／Southern印迹法分析发现,83％的转基因植株含有所有3个外源基因。Western印迹法分析发现79％的含gna基因的转基因植株以不同水平表达GNA。遗传分析证实外源基因在转基因植株后代中以孟德尔方式遗传。从其R1代亲本为孟德尔3：1方式遗传的R2代中,鉴定出2个含有所有3个外源基因的独立转基因植株纯系。这些纯系具有相似的外源基因表达量。褐飞虱喂养试验表明,这些纯系对褐飞虱具有显著的抑制作用。这些褐飞虱抗性提高的转基因纯系将应用于水稻抗虫育种中。实验证明,通过遗传转化和筛选可获得含在农业上有应用价值基因的转基因水稻纯系。     

转多个抗真菌蛋白基因水稻植株的获得及其抗稻瘟病菌的初步研究

用基因枪法将水稻碱性几丁质酶（ＲＣ２４）基因、苜蓿葡聚糖酶（βＧｌｕ）基因、大麦核糖体失活蛋白（ＢＲＩＰ）基因和潮霉素（ｈｐｔ）基因同时导入籼稻品种（七丝软占）中,获得了７个潮霉素抗性再生系,Ｓｏｕｔｈｅｒｎｂｌｏｔ证明２～３个抗真菌蛋白基因已整合到水稻基因组中．初步抗性鉴定表明Ｒ０代转基因水稻植株对稻瘟病菌的抗性有所提高     

转基因技术及其在水稻育种中的研究进展

以载体介导、基因直接导入和种质系统3类基因转化体系分别介绍了常用的几种转基因技术—农杆菌转化法、基因枪法、PEG介导法、电击法、花粉管通道法和浸泡转化法的原理及其在水稻育种上的最新研究进展,比较了各种技术的特点。最后对转基因技术在水稻育种上的前景作了展望。     

通过遗传转化获得含多基因的水稻转基因纯合植株

利用基因枪法将含有4个不同基因的3个质粒共转化由粳稻品种鄂宜105号和鄂晚5号种子胚诱导的愈伤组织（5－10d龄）。从轰击的986块愈合组织中共再生出169株独立的转基因水稻植株（转化率为17％）。PCR／Southern blot分析显示70％以上的转基因植株含有所有4个基因。GUS组织化学分析、Western blot和或RT－PCR分析表明所有4个基因的共表达率为70％。未观察到任何质粒在整合中存在优势,转基因拷贝数也与基因表达量无关。遗传分析证实外源基因在后代植株中大多以孟德尔方式遗传。从其R1代为3：1孟德尔方式遗传的后代R2代植株中,鉴定含有3个或4个不同基因的转基因纯合植株系。PCR／Southern blot分析证实了这些转基因纯合植株系。这些系的植株具有相似的外源基因表达量。我们证实通过基因枪介导的共转化,结合常规育种方法筛选可以获得含多基因的转基因水稻纯合植株。这项技术为利用基因同时改良作用多个性状提供了一种途径。     

一个水稻蛋白激酶的过表达分析及表达调控

对一个预测的具有光反应活性的丝氨酸/苏氨酸蛋白激酶STK进行过表达研究,构建了STK过表达载体,并用农杆菌介导的方法进行遗传转化,获得T0代转基因植株.对转基因阳性植株进行表达分析的结果表明,转基因植株STK基因的表达量显著高于对照,说明目的基因得到了过表达.另外,利用获得的过表达转基因植株对STK调控水稻中光周期相关的开花基因Hd1和Hd3a的表达进行分析,结果表明,Hd1及Hd3a的表达在转基因植株中明显上调,表明该蛋白激酶的基因可能位于Hd1和Hd3a上游,对于Hd1和Hd3a的表达具有重要的调控作用.     

抗除草剂基因atzA在转基因水稻中的遗传

研究了细菌阿特拉津氯水解酶基因atzA在转基因水稻AA269株系自交和回交后代中的遗传规律．结果表明：atzA基因作为一个显性遗传位点遵循孟德尔遗传分离规律．除草剂抗性鉴定结果表明,在T1代水稻群体中除草剂抗性和敏感株数遵循3：1的分离规律,T2代稳定纯合;T2～T7自交世代的转基因植株均保持稳定的除草剂抗性．在BC1～BC3代群体中除草剂抗性和敏感株数遵循1：1的分离规律,BC群体自交的BCF2群体中遵循3：1分离规律．分别对AA269／9311的B3F1和B2F2群体中的一个株系进行了PCR分析,PCR阳性株与阴性株的分离比例分别符合1：1和3：1,与除草剂抗性鉴定结果一致．对T7代群体中部分单株的Southern杂交和RT—PCR分析表明,atzA基因已稳定遗传至T7代,并得到表达．     

Transformation of japonica rice with RHL gene and salt tolerance of the transgenic rice plant

Overexpression of the yeast HAL2 gene increases salt tolerance of yeast and plant. Rice HAL2-like (RHL) gene was introduced into a japonica rice cultivar HJ19 with Agrobacterium tumefaciens-mediated transformation. Transgenic plants in R0 generation were selected on the principle of GUS-positive, RHL gene PCR-positive and normal growth. Hygromycin-resistant plants of some transgenic lines in R1 generation increased salt tolerance during the seedling and booting stage, being less damaged in the cytomembrane and stronger in leaf tissue viability under salt stress during booting period. Southern analysis of transgenic lines tolerant to salt in R1 generation showed that the RHL gene expression cassette had been successfully integrated into rice genome. Moreover, gene engineering breeding methodology and really salt-tolerant rice cultivar were discussed.     

Rice transformation with a senescence-inhibition chimeric gene

A senescence-inhibition chimeric gene containing the specific promoter of SAG₁₂ and IPT gene was transferred into rice with the biolistic method. Results of PCR, Dot blotting and Southern blotting indicated that the chimeric gene had been integrated into rice genome. Analyses of GUS activity and cytokinin content in transgenic plants of rice and the observation of T₁ generation plant at grain formation stage indicated that the foreign gene was expressed.     

外源基因（bar）在转基因燕麦（AvenasativaL.）后代中的遗传与表达

通过微弹轰击获得的含有ｂａｒ基因的转基因燕麦植株自交产生转基因后代。利用ＰＣＲ方法检测ｂａｒ基因在后代中的传递情况。     

Transformation of japonica rice with<Emphasis Type="Italic">RHL</Emphasis> gene and salt tolerance of the transgenic rice plant

Overexpression of the yeastHAL2 gene increases salt tolerance of yeast and plant. RiceHAL2-like (RHL) gene was introduced into ajaponica rice cultivar HJ19 withAgrobacterium tumefaciens-mediated transformation. Transgenic plants in R₀ generation were selected on the principle of GUS-positive,RHL gene PCR-positive and normal growth. Hygromycin-resistant plants of some transgenic lines in R₁ generation increased salt tolerance during the seedling and booting stage, being less damaged in the cytomembrane and stronger in leaf tissue viability under salt stress during booting period. Southern analysis of transgenic lines tolerant to salt in R₁ generation showed that theRHL gene expression cassette had been successfully integrated into rice genome. Moreover, gene engineering breeding methodology and really salt-tolerant rice cultivar were discussed.     

转基因水稻与野生稻（Oryza nivara Sharma et Shastry）杂交后代种子萌发率

种子休眠性是野生植物重要的适应性状,通过种子萌发实验可以分析种子休眠性强弱.通过人工杂交获得转抗虫基因栽培稻与一年生普通野生稻三种组合的杂种,对杂种的F3代和F4代种子采用直接萌发、打破休眠后萌发、埋土15 d和30 d 4种不同处理来检测种子活力和萌发率.结果显示转基因杂种后代种子表现出较强的休眠性,转基因对种子活力和休眠性没有明显的影响,种子休眠性有随种子世代的增加而逐渐减弱的趋势,提示水稻转基因逃逸后有在野生稻群体中宿存和扩散的可能性,但这种可能性可能会随世代增加而下降.这为进一步研究水稻转基因逃逸风险提供了参考.     

Salt tolerance of transgenic rice (Oryza sativa L.) with mtlD gene and gutD gene

Southern blot analysis indicated that mtlD gene (encoding mannitol-1-phosphate dehydrogenase) and gutD gene (encoding glucitol-6-phosphate dehydrogenase) had been integrated into the rice genome mediated by Agrobacterium tumefaciens LBA4404(pBIGM). The expression of the above two genes in transgenic rice plants was demonstrated by Northern blot analysis and enzymatic activity assay. Analysis of sugar alcohol showed that transgenic rice plants could produce and accumulate mannitol and sorbitol. The salt tolerance of transgenic plants was much higher than that of their controls.     

Introduction of antisense waxy gene into main parent lines of indica hybrid rice

Amylose content in rice endosperm is one of thekey determinants of rice eating and cooking quality, and thepoor quality of indica hybrid rice is closely related to thehigh amylose level in rice grains. In order to improve thegrain quality of the indica hybrid rice by genetic engineering,an antisense fragment of rice waxy gene, driven by theintroduced into three major parent lines of indica hybrid rice,all contain a high amylose level in the grains, via Agrobacte-rium, and more than 100 hygromycin-resistant plants wereregenerated. The analysis of PCR amplification and South-ern blots indicated that the T-DNA containing the antisensewaxy gene had been integrated into the genome of transgenicrice plants. Most of the primary transgenic rice plants grewnormally, and the mature seeds from these transgenic plantswere performed for analysis of the amylose content. Theresults showed that the amylose content in the endosperm ofsome grains was reduced and the lowest reached 7.02% inone homozygous transgenic line, 72.4% lower than that ofthe wild type. The influence of the altered amylose contenton the gelatinization temperature and gel consistency wasalso observed in several homozygous transgenic rice plants.     

Salt tolerance of transgenic rice (Oryza sativa L.) withmtlD gene andgutD gene

Southern blot analysis indicated thatmtlD gene (encoding mannitol-1-phosphate dehydrogenase) andgutD gene (encoding glucitol-6-phosphate dehydrogenase) had been integrated into the rice genome mediated byAgrobacterium tumefaciens LBA4404(pBIGM). The expression of the above two genes in transgenic rice plants was demonstrated by Northern blot analysis and enzymatic activity assay. Analysis of sugar alcohol showed that transgenic rice plants could produce and accumulate mannitol and sorbitol. The salt tolerance of transgenic plants was much higher than that of their controls.     

抗除草剂旱稻转基因植株的获得

以旱稻丹粳旱５－５５、６－２４、６－３７的悬浮细胞及未成熟胚为受体材料，采用基因枪法将含ＢＡＲ基因的ｐＤＭ３０２质粒ＤＮＡ导入旱稻细胞中，经ＰＰＴ筛选获得抗性愈伤组织，筛选出的愈伤组织在分化培养基上再生出完整的旱稻转基因植株。Ｓｏｕｔｈｅｒｎ分子杂交分析表明，外源ＢＡＲ基因已整合到水稻基因组内，抗除草剂试验结果表明，转化植株对０．０１％Ｂａｓｔａ（有效成分为ＰＰＴ）有一定程度的抗性，说明外源ＢＡＲ     

Antisense expression of a rice cellular apoptosis susceptibility gene (OsCAS) alters the height of transgenic rice

Cellular apoptosis susceptibility (CAS) gene plays important roles in mitosis, development and export of importin α from the nucleus, but its function in plant is unknown. In this study, a rice CAS ortholog (OsCAS), which encodes a predicted protein of 983 amino acids with 62% similarity to human CAS, was identified. DNA gel blot analysis revealed a single copy of OsCAS in the rice genome. A 973 bp fragment at the 3′ end of OsCAS cDNA was cloned from rice cDNA library and transferred into rice in the antisense direction under the control of CaMV 35S promoter via Agrobacterium-mediated transformation method, 105 transgenic lines were obtained. Expression of OsCAS was suppressed in the antisense transgenic lines as revealed by semi-quantitative RT-PCR. The antisense transgenic lines showed dwarf phenotypes. The results indicated that OsCAS was involved in culm development of rice.