【作者】 黄科；

【导师】 曹家树；

【作者基本信息】 浙江大学 ， 蔬菜学， 2004， 博士

【摘要】 芸薹属（Brassica）植物是杂种优势利用最为普遍的一类作物，其雄性不育的机理以及雄性不育系选育的研究一直深受研究人员的重视。我们前一阶段利用mRNA差异显示技术从白菜（B．campestris L．ssp．chinensis（L．）Makino var．communis Tsen et Lee）核雄性不育两用系中分离得到了一个与白菜核雄性不育相关的编码细胞色素P450基因CYP86MF。采用反义技术将可育株高表达的CYP86MF基因中约460 bp的片段导入正常可育白菜和菜心（B．campestris ssp．chinensis var．parachinensis（Bailey） Tsen et Lee）中，得到了130多株转化雄性不育植株。为进一步了解本实验室获得的CYP86MF基因在芸薹属其它作物上的生物学功能，在建立了青花菜和芥蓝的高效再生体系的基础上，我们将上述反义片段导入正常可育的青花菜（B．oleracea L．var．italica P．）和芥蓝（B．oleracea var．alboglabra）中，得到了260多株转化植株。转基因青花菜雄蕊表现出明显的褐化现象，花粉少，转基因芥蓝花器官则没有明显的特征，只是相对于对照植株而言，花丝略微短小。转基因植株的花粉均不能正常萌发。我们分析了转基因不育植株与正常可育植株CYP86MF基因的表达特征，对转基因植株及其对照株之间的小孢子的形态进行了观察，比较了它们之间蛋白质表达的差异，从而为探讨CYP86MF基因在芸薹属植物小孢子发育过程中的作用机制提供了一些信息。主要研究结果如下： （1）采用正交设计方法，通过不同浓度的激素配比、AgNO₃浓度、蔗糖浓度以及不同外植体的筛选，提出了青花菜离体培养再生频率较高的培养基配方(MS+NAA 0.02 mg·L^-1+BA 4mg·L^-1+2％蔗糖+0.8％琼脂)，最适宜的外植体类型为下胚轴，再生频率达到100％。再生植株移栽的成活率达到98％以上。该培养基对青花菜的不同品种有着较广的适应性。 （2）采用正交设计方法，通过不同浓度的激素配比、AgNO₃浓度、蔗糖浓度以及不同外植体的筛选，提出了芥蓝离体培养再生频率较高的培养基配方(MS+NAA0.03 mg·L^-1+BA 2 mg·L^-1+3％蔗糖+AgNO₃ 10.5 mg·L^-1+0.8％琼脂)，最适宜的外植体类型为下胚轴，再生频率达到97.5％。再生植株移栽的成活率达到90％以上。该培养基对芥蓝的不同品种有着较广的适应性。 （3）为了解不同培养基诱导愈伤组织和不定芽的差异机制，采用cDNA-AFLP和双向电泳技术分别对分化培养基和不分化培养基上的外植体进行了分析，结果表明，两种外植体住表达水平和翻译水平存在差异，cDNA-AFLP特异条带主要出现在分化培养基的外植体上，且主要集中在200～600 bp之间；双向电泳结果发现差异蛋白多分布在PI 5～7，分子量40～70 kD之间。 （4）采用正交旋转组合设计方法，通过对青花菜和芥蓝Kan筛选浓度、预培养时间、共培养时间、农杆菌感染时间和抑菌剂浓度的筛选，建立了青花菜和芥蓝高效遗传转化体系。具体操 摘要作为:将卜胚轴外植体在预培养基上预培养3d（青花菜）或Zd（芥蓝）后，用LBA4404（pBI35s一AMF）感染8 min，在灭菌滤纸上吸干菌液，置于共培养培养基上培养Zd，随后将外植体转入含5mg’L一’Kan的选择分化培养基上诱导不定芽，3周转瓶一次，当抗性幼苗长至2一3。m时，在愈伤组织处切卜幼苗，置于生根培养基上诱导不定根，20d左右不定根长成后，开瓶炼苗，继而移栽至营养十中，按止常栽培措施进行后期管理。 （5）按己建立的转化体系，获得了260多株青花菜和芥蓝的抗卜那霉素苗。共对%株KanR ‘新绿’青花菜和4株KanR‘中花’芥蓝进行了PcR检测，其中青花菜有83株呈阳性，阳性率为86.46%，‘中花’芥蓝有2株呈阳性，阳性率为50%:Southem印迹杂交检测结果显示，16个PCR检测成阳性的青花菜样品中，有12个样品在Southem印迹杂交中有阳性信号，阳性率为75%，在2个PCR检测成阳性的芥蓝样品中，有1个样品在southem印迹杂交中有阳性信号，阳性率为50%。随后以转基因‘新绿’青花菜和‘中花’芥蓝的对照株为阴性对照进行Northern印迹杂交，结果显示，转基因‘新绿’青花菜和‘中花’芥蓝植株中CYP86九fF基因的表达丰度均显著低于对照株，说明CYP86人石F基因在甘蓝类作物中的确有表达:转基因‘新绿’青花菜和 ‘中花’芥蓝中反义C理86几护基因片段进行了转录，并部分抑制了CYP86人办，基因转录的mRNA的活性。 （6）在获得的260多株转基因植株中，转基因青花菜雄蕊表现出明显的褐化现象，花粉少，转基因芥蓝花器官则没有明显的特征，只是相对于对照植株而言，花丝略微短小。转基因植株的花粉均不能正常萌发。比较分析了两者花粉的电镜扫描形态差异，结果表明，转基因植株的花粉粒表现出畸形，畸形率达到90%以上。说明反义CYPS占A石F基因片段的转化对‘新绿’青花菜的花器结构以及小抱子的发生过程有一定的影响，对‘中花’芥蓝的花器结构没有明显的影响，但对小抱子的发生有一定的影响。 （7）为进一步了解C)沪86A工F基因在蛋自质水平上的差异，采用双向电泳技术分别对自菜核雄性不育两用系可育株和不育株材料、转基因青花菜?更多还原

【Abstract】 Brassica crops is a kind of crops that the most successful in utilizing of heterosis in China. Much attention was paid to research on the plant breeding of the male sterile line and the basis for application in Brassica crops. A cytochrome P450 gene CYP86MF was obtained in floral bud of Chinese cabbage pak-choi (B. campestris ssp. chinensis (L.) Makino var. communis Tsen et Lee) between A/B line by mRNA differential display PCR technology (DD-PCR) and rapid amplification of cDNA ends (RACE) technology. Chinese cabbage-pak-choi and flowering Chinese cabbage (B. campestris ssp. chinensis var. parachinensis (Bailey) Tsen et Lee) were inoculated with Agrobacterium tumefaciens strain LBA4404 containing pBI35S-AMF carrying antisense gene CYP86MF. More than 130 plantlets KanR seedlings were obtained. In order to find out the function of gene CYP86MF in other Brsssica crops, we study further in this paper. After the efficient shoot regeneration system of B. oleracea L. var. italica P. and B. oleracea var. alboglabra were established and using hypocotyl as explants inoculated with the antisense gene. More than 260 plantlets KanR seedlings were obtained after we constructed an efficient genetic transformation system. The stamen of transgenic B. oleracea var. italica. were browned and there are little pollen in anther, but the stamen of transgenic B. oleracea var. alboglabra were natural. The pollen of transgenic couldn’t germinate normally. Therefore, the expressing characteristics of gene CYP86MF were analyzed by Northern hybridization in transgenic plant and non-transgenic plant, the morphology of microspore were observed in transgenic plant and non-transgenic plant, the difference protein between transgenic and un-transgenic plant were observed. The primary results obtained from the research provide some clues for the study on the biological function of gene CYP86MF and molecular mechanism of the male sterility. The main results are as follow:(1) the efficient plant regeneration of B. oleracea var. italica was established by orhtogonalty design, optimized the concentration of NAA, BA, AgNO3 and sucrose, the type of explant was selected. Results showed that the regeneration frequency reached 100 % in this medium with MS + NAA 0.02 mg L-1 + BA 4 mg-L-1 + 2 % sucrose + 0.8 % agar, the best explant is hypocotyls, and the livability of regeneration plantlet is 98%. This formula is fit for other cultivars in B. oleracea var. italica.(2) the efficient plant regeneration of B. oleracea var. alboglabra was established by orhtogonalty design, optimized the concentration of NAA, BA, AgNO3 and sucrose, the type of explant was selected. Results showed that the regeneration frequency reached 97.5%in this medium with MS + NAA 0.03mg L-1 + BA 2mg L-1 + 3 % sucrose + AgNO3 10.5 mg L-1 + 0.8 % agar, and the best explant is hypocotyls, and the livability of regeneration plantlet is 90%. This formula is fit for other cultivars in B. oleracea var. alboglabra.(3) the difference of explant on differentiation medium and un-differentiation medium were analysised by cDNA-AFLP and two-dimension electrophoresis. The results showed that there are 13 bands which distributed from 200 bp to 600 bp, and the difference protein distributed PI 5 ~ 7, MW 40 ~ 70 kD.(4) the efficient gene transformation system of B. oleracea var. italica and B. oleracea var. alboglabra were established by orhtogonalty rotation design, optimized the pre-culture time, co-culture time, infective time, the concentration of Kan and Amp. The procedure was as follow: pre-cultured 3 days {B. oleracea var. italica) or 2 days (B. oleracea var. alboglabra), A. tumefaciens strain LBA4404 infected 8 min, co-cultured days. Then the co-cultured explants were inoculated into the regeneration medium containing the 5 mg-L"1 Kan to obtain the KanR shoot, and the explants were exchanged to fresh medium per 3 weeks. When the KanR seedlings grew to 2 ~ 3 cm, cut them and inoculated into the root induced medium. Roots were induced about 20 days, then opened the plastic fi更多还原