抗菌肽基因转化大白菜获得抗病转基因植株及稳定遗传

软腐病是大白菜( Brassica pekinensis Rupr.)的三大病害之一.抗菌肽对软腐病菌有很强的杀伤作用.建立了根癌土壤杆菌( Agrobacterium tumefaciens ) EHA105(pMOG410)工程菌的高频转化载体系统,将抗菌肽基因导入目前推广种植的大白菜AB-81自交系,获得了转基因植株.PCR及 Southern blotting分子杂交鉴定表明抗菌肽基因已整合到白菜基因组.转基因植株提取液的体外抑菌实验、试管苗及盆栽转基因植株的病原菌接种抗病测试结果表明转基因植株具有明显的抗病特性,并且能稳定遗传,转基因植株R1自交分离比为3∶1,R5的转基因植株保持抗Km和抗病特性,可望以其为亲本选育出大白菜抗软腐病的新品种.     

大白菜生物技术研究进展

综述了国内外在大白菜(Brassica rapa ssp.pekinensis)单倍体培养、基因工程和分子标记方面的最新研究成果.重点讨论了大白菜小孢子培养的影响因素、取材要点、培养条件、植株再生和倍性鉴定以及大白菜的转基因载体选择、受体系统建立、遗传转化与转基因植株鉴定等.对小孢子培养及转基因研究的原理、方法和关键技术进行了总结.展望了大白菜生物技术研究的应用前景.     

Cloning and Characterization of the Microspore Development-Related Gene BcMF2 in Chinese Cabbage Pak-Choi （Brassica campestris L. ssp. chinensis Makino）

For the sake of providing some important information relevant to the study of the molecular mechanism of genic male sterility in plants, gene differential expression in flower buds at different developmental stages, as well as in rosette leaves, florescence leaves, and scapes was analyzed using cDNA amplified fragment length polymorphism (cDNA-AFLP) in the genic male sterile A and fertile B line of Chinese cabbage pak-choi. Following amplification of 125 pairs of primer combinations, 11 differential fragments were obtained, of which eight were from the B line and the other three were from the A line. Of 11 differential fragments, four were verified by Northern hybridization that were expressed preferentially in fertile flower buds. Results of GenBank BLAST showed that one fragment was with unknown function, whereas the other fragments have strong nucleotide sequence similarities with the polygalacturonase (PG) gene, the pectinesterase (PE) gene, and the polygalacturonase inhibitory protein (PGIP4) gene. Only fulllength cDNA from the differential fragment BcMF-A 18T 16-1 was amplified by rapid amplification of cDNA ends (RACE) and Northern analysis showed that this fragment was expressed only in medium and largesized flower buds of the B line. The full-length cDNA, designated as BcMF2 (Brassica campestris Male Fertile 2), was 1 485 bp long and was composed ofa 1 263-bp open reading frame, which had 83% nucleotide similarity to a PG gene from Arabidopsis encoding polygalacturonase. Analysis of the basic structure of the protein revealed that it had one polygalacturonase active site (RVTCGPGHGLSVGS) at 256th site of amino acids and was classified as being a member of family 28 of the glycosyl hydrolases. The role of the BcMF2gene on microspore development is discussed in the present paper.     

大白菜细胞核复等位雄性不育基因定位

以大白菜不育材料"939A"为母本,携带恢复基因的材料YDQ56A为父本构建隐性雄性不育F1S4分离群体,利用混合分组分析法(BSA)对不育基因初定位;同时以"939A"为母本,携带保持基因的材料yellow sason为父本构建显性雄性不育F2分离群体,对不育基因进行精细定位。结果在F1S4群体中获得与不育基因连锁的标记2个,A08_1900和Br ID111035,与不育基因分别相距8.8c M和2.5c M,物理距离为804.1kb;F2群体中不育单株与可育单株分离比符合3∶1,不育基因表现为显性。通过标记验证,F2和F1S4两个群体定位结果一致,均位于A08染色体末端,通过新标记的筛选获得不育基因两侧紧密连锁的标记Br19470306和Br19675586,与不育基因分别相距1.6c M和2.4c M,物理距离205.28kb,其中包含58个基因。该结果为大白菜雄性不育系的利用以及转育奠定了基础。     

一个大白菜结球—抽薹模型及其特性

用大白菜形态发生上的源库关系原理,建立了一个二元二次微分方程组模型．通过对方程组的化简证明,在大白菜的结球—抽薹上,球叶的肥大与花茎的发生为一养分竞争关系．     

大白菜显性不育基因向白菜型油菜上转育的遗传研究

利用大白菜的显性核不育基因向白菜型油菜上转育,成功地育成了白菜油菜３种不育系。其配合力测定的总趋势,甲型不育〉全不育〉乙型不育,经遗传分析,选育,找到了上位显性核互作恢复基因（Ｍｓ）,它对核不育基因（Ｓｐ）具有上位恢复作用,育性是２对显性核不育基因（Ｓｐ）和上位显性核互作恢复基因（Ｍｓ）控制的。并弄清了其相应的遗传模式。     

大白菜显性核不育基因向白菜型油菜上转育的遗传研究

利用大白菜的显性核不育基因向白菜型油菜上转育,成功地育成了白菜型油菜３种不育系。其配合力测定的总趋势：甲型不育＞全不育＞乙型不育。经遗传分析、选育,找到了上位显性核互作恢复基因（Ｍｓ）,它对核不育基因（Ｓｐ）具有上位恢复作用。育性是由２对显性核不育基因（Ｓｐ－）和上位显性核互作恢复基因（Ｍｓ－）控制的。并弄清了其相应遗传模式。     

转双价抗虫基因大白菜新种质的获得及其抗虫性分析

采用超声波辅助花粉介导方法,将双价抗虫基因BmkIT-Chitinase导入早熟型大白菜自交系20-19-3,最终获得了5个转基因大白菜优良自交系纯合株系Z1-5、Z2-7、Z9-6、Z11-6和Z20-13;以转基因大白菜株系和非转基因对照植株为材料,对BmkIT-Chitinase基因在大白菜中的遗传规律、基因表达及抗虫性进行进一步分析。结果显示:(1)转化株后代多代(T_1~T_4)PCR、Southern blotting等分子跟踪检测表明,目的基因已成功导入受体植株,且能够稳定遗传;用该转基因方法对大白菜进行基因转化所获得的转基因植株分析显示,外源基因多数以多拷贝形式整合于核基因组,少部分外源基因以单拷贝形式整合。(2)Elisa分析结果证明,所导入的外源基因可高效表达,T4代株系新鲜叶片中表达产物量最高达到0.069μg·g~(-1)左右。(3)转基因株系田间抗虫性统计分析表明,转化株系与对照在抗虫性方面有显著差异,其对小菜蛾及菜青虫抗性普遍提高2~3级。研究认为,转BmkIT-Chitinase基因大白菜中BmkIT-Chitinase基因的表达可有效提高大白菜的抗虫性。     

菌体脂多糖在大白菜软腐欧文氏菌对寄主根表吸附中的作用

用两种方法分别洗除菌体胞外多糖(EPS)和脂多糖(LPS),经在大白菜幼苗上测定表明,菌体洗除LPS后对寄主根表的吸附能力明显减弱,吸附菌量随无LPS的菌体在混合接种体中比例的增高而降低,在无LPS的菌体接种体中加入LPS提取物,可使吸附能力恢复到正常水平;平行测定的EPS不发生上述影响。用EPS和LPS对根表进行预处理,均可明显抑制完整菌体(不洗,含EPS和LPS)和无EPS的菌体对根表的吸附,但只有LPS预处理使无LPS的菌体吸附量提高2—3倍。用大白菜对该菌的专化性细菌凝集素Agin—SD60所作的菌体凝集试验表明,菌体洗除LPS后不再被 Agin—SD60所凝集。根据以上结果认为,菌体脂多糖在大白菜软腐欧文氏菌与寄主的接触识别中可作为细菌识别子起作用。     

大白菜(Brassica pekinensis)与甘蓝(B.oleracea)的种间试管受精

通过大白菜胚珠与甘蓝花粉的试管授粉和受精得到了杂种种子。杂交一代的植株形态和花粉发育情况及根尖细胞染色体数均表明为种间杂种。杂种植株具有父母本的特征,某些性状介于双亲之间;花粉粒发育不正常;根尖细胞染色体数为2n=19。本研究为其他十字花科植物的远缘杂交中克服不亲和性提供了依据。