【作者】 周瑞金；

【导师】 杜国强；

【作者基本信息】 河北农业大学 ， 果树学， 2007， 博士

【摘要】 苹果是世界上最重要的果树树种之一。苹果转基因研究开始于1989年，随后相继有苹果砧木、品种基因转化成功报道，但研究多集中于基因转化，关于外源基因在转化植株中的表达、特性以及外源基因在转化系中的遗传规律等研究尚少，开展此方面研究将加速转基因苹果在生产中的应用和推广。本试验以转豇豆胰蛋白酶抑制剂(Cowpea Trypsin Inhibitor，CpTI)基因苹果(Malus domestica Borkh.)(包括皇家嘎拉、王林、乔纳金和红富士60个株系)为试材，应用PCR、RT-PCR、荧光原位杂交、室内虫试等方法，研究了外源CpTI基因在苹果转化组培苗中DNA、RNA和蛋白等水平的表达；应用转基因花粉培养、果实蛋白毛细管区带电泳等方法，研究了转基因苹果花粉和果实的特性；通过杂交授粉试验，研究了外源基因在转基因苹果中的遗传规律；通过试管微嫁接方法，研究了标记基因在苹果砧穗间的传导性。主要研究结果如下：1在60个常规继代培养6～8年的转CpTI基因苹果转化株系中均可检测出CpTI特异基因片断；除2个转化株系在50 mg／L卡那霉素浓度下出现花叶现象，表现缺乏卡那霉素抗性外，其他株系在含相同浓度卡那霉素的培养基中生长正常。2通过对FISH的相关技术参数研究，初步建立了苹果染色体制备和荧光原位杂交技术体系。以CpTI基因片断为探针，利用该体系在苹果细胞核上成功检出了荧光杂交信号，表明外源基因整合到了转基因苹果的基因组中。3部分转CpTI基因苹果株系有较强的杀虫和抑虫作用。60个转化株系中有2个株系虫试校正死亡率为100％；28个株系校正死亡率为正值，其中11个株系校正死亡率大于等于50％，经秩合检验分析，有4个转化株系的虫重与对照虫重相比差异显著，表明其有明显抑制幼虫生长发育的作用。4喂食转CpTI基因苹果组培苗叶片的棉蛉虫体内类胰凝乳蛋白酶活性变化结果表明：不同转基因株系对虫体内类胰凝乳蛋白酶活性的抑制作用存在差异，分别表现为抑制能力较强、较弱或没有抑制能力。其中有9个株系能显著抑制棉蛉虫幼虫体内类胰凝乳蛋白酶活力，表明这些株系具有较好的抑制幼虫生长发育的作用。5对60个转化株系苹果组培苗进行RT-PCR检测，结果表明转入的外源CpTI基因在43个株系中得到了较高水平的表达，在17个株系中表达强度低。外源基因在转化株系(嘎拉32)中高效表达，经RT-PCR扩增，片断回收，克隆，测序，外源基因在转基因植株中核苷酸表达与Phaseolus vulgaris trypsin proteinase inhibitor gene和Tieganqing Irypsin inhibitor(TI)gene有100％同源性，蛋白表达与proteinase inhibitor-cowpea有100％同源性。6优化的适宜苹果叶片蛋白质毛细管电泳程序为：采用改良丙酮沉淀法提取蛋白质，电泳过程中温度为25℃，电压为20 kV，0.5 psi进样5 s，电泳时间为17 min，检测波长在280 nm处的蛋白质区带。电泳结果表明：与对照相比有4个嘎拉转化株系在第7.65 min分别出现一条特异吸收峰带。7转基因苹果花粉离体萌发率(24.47％)低于对照(62.05％)，但转基因花粉对卡那霉素的抗性高于对照。扫描电镜观察未发现外源基因的导入对苹果花粉粒形态、大小、纹饰等方面产生明显的影响。8转基因嘎拉大部分果实中可以检测到nptⅡ酶活性，但不同果实中的nptⅡ酶活性强度有差异，其中nptⅡ酶活性较强的占71.43％，较弱的占21.43％，检测不到的占7.14％；果实蛋白毛细管区带电泳检测结果显示，转基因苹果嘎拉果实中蛋白种类少于对照。9以嘎拉转化株系与未转化富士苹果进行杂交，对果实种胚培养并利用卡那霉素抗性和PCR检测外源基因存在状况，结果表明杂交实生后代外源基因分离比例1：1，符合由一对显性基因控制的遗传性状的自由分离规律，表明外源基因为一显性基因，呈单点显性遗传。10转基因组培苗微嫁接接穗以选用继代培养30 d左右，带有2～4片叶片的新梢为宜；适当提高BA浓度有利于提高嫁接成活率，其中以MS+BA 1.0 mg／L+NAA 0.05 mg／L培养基中嫁接成活率最高，达83.3％。以转基因嘎拉、王林、乔纳金和富士作为接穗研究结果表明，不同品种之间嫁接成活率差异不显著。外源nptⅡ基因只在转化植株体内表达，未通过微嫁接在砧穗间产生基因效应。更多还原

【Abstract】 Apple is one of the most important fruit trees in the world. The research on genetransformation in apple has been conducting since 1989. Then some of the foreign geneswere transferred into apple rootstocks and cultivars successfully. But the studies weremainly focused on the process of the gene transformation, and few studies on expression,characteristics and inheritance of the exogenous genes were found. The study in theaspects of these issues would benefit to the application of gene transformation in appleand others.The transgenic lines of apple （Malus domestica Borkh.） carrying exogenousCowpea Trypsin Inhibitor （CpTI） gene including Royal Gala, Orin, Jonagold and Fujiwere used to study the expression of the CpTI gene in DNA, RNA and protein levels byusing the techniques of PCR, RT-PCR, FISH, electrophoresis and feeding the Helicoverpaarmigera larvae with the leaves. Meanwhile, the characteristics of the pollen and fruitsfrom the transgenic lines were evaluated. The inheritance of the CpTI gene was studiedby the reciprocal cross. The conducting effect of the report gene （NeomycinphosphotransferaseⅡ, NptⅡ） between the rootstocks and scions was studied by usingmicro-grafting. The main results were as follows:1. The presence of exogenous CpTI and nptⅡgenes in transgenic lines conservedfor 6～8 years by subculture at normal temperature were studied by the techniques ofPCR analysis and Kanamycin resistance evaluation. The PCR analysis results showedthat all transgenic lines could generate the expectable band. The leaves of two linespresented yellow when the plantlets cultured on the medium with 50 mg/L Kanamycin,which indicated the lack of the Kanamycin resistance, and the others could grownormally.2. The preliminary FISH technique system on apple was set up. The signals wereobserved successfully on the cell nucleus of the transgenic lines by using the probe of CpTI gene.3. The feeding experiment showed that some transgenic lines had the effect onrestricting the larvae growth or killing the larvae. There were two lines had the strongestaffection by killing the larvae of 100%. Twenty eight lines showed positive value of themodified death rate. And the modified death rate of the eleven lines was higher than orequal to 50%. It indicated that those lines had the ability to restrict the growth of the larvaeor kill the larvae. The weight of the larvae that fed by the leave of four lines showedsignificant difference from the control.4. The activity of the midgut chymotrypsin-like enzyme of the larvae after fed by theleave varied from the transgenic lines. The activity was significant difference from thecontrol when the larvae were fed by the leave collected from nine lines. That indicated thoselines had certain capacity of restraining the growth and development of the insect.5. Testing of the expression of exogenous CpTI by RT-PCR analysis in sixty linescultured in vitro indicated that there were high expression in forty three lines, and theexpectable band in seventeen samples was dim. The CpTI gene fragments fromtransgenic line Gala 32 was amplified, recoveryed, cloned and sequenced. The BLASTresult of the nucleotide acid sequence showed that the obtained gene fragment was highlyhomologous with phaseolus vulgaris trypsin proteinase inhibitor gene and tieganqingtrypsin inhibitor （TI） gene, and the degree ranged was 100%. The protein analysisshowed that the obtained gene fragment was highly homologous with proteinaseinhibitor-cowpea.6. The optimal protocol of Capillary Zone Electrophoresis （CZE） analysis for appleleaves was that: extracting the protein form leave by using improved ace-sediment method;setting the voltage as 20kV and the temperature as 25℃; setting the isoelectric focusingtime as 17 minutes and the wavelength as 280 nm. The result of CZE showed that at the7.65 minutes the exceptional protein brands were founded in the four transgenic lines ofsixty.7. The in vitro pollen germination rate for transgenic apple was 24.47% at 12 hours,lower than that of the control （62.05%）. But the exogenous CpTI gene could increase theKanamycin resistance of pollen. The shape, size and ornamentation of pollen grains observedby SEM showed no difference between the transgenic apple and non-transgenic controlapple.8. Neomycin phosphotransferase （nptⅡ） activities were detected in the most transgenicapple fruits, but the activities varied from the fruits with strong in 71.43%, weak in 21.43%,and no activity in 7.14%. The result of CZE for the fruits showed that the types of protein in transgenic Gala were few than those in non-transgenic control Gala.9. The embryos collected from the fruits by crossing between transgenic andnon-transgenic Gala and Fuji were cultured in vitro for new plantlet’s generation. Thenthe Kanamycin resistance ability of the plantlets was evaluated. The inheritance patternsof F₁ plants showed about 1:1 segregation ratio, in accordance with Mendelian rule ofdominant gene with singal locus.10. For micro-grafting, the higher survival grafting rate was obtained when the plantletsthat subcultured for 30 days with vigorous adventitious shoots and the scion with 2～4leaves were used. The moderate increasing of the concentration of BA in the medium couldincrease the survival grafting rate. The suitable medium for micro-grafting in vitro wasMS+BA1.0mg/L+NAA0.05mg/L with a highest grafting rate of 83.3%. Meanwhile, noeffect was found on the survival grafting rate due to the exogenous gene transformation inapple. The exogenous nptⅡgene was only expressed in the transgenic explants and noconductivity was found between the scions and rootstocks by micro-grafting.更多还原