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【目的】探讨花药绒毡层特异启动子BcA9调控的反义BcMF12基因对白菜（Brassica campestris L. ssp. chinensis, syn. B. rapa L. ssp. chinensis）基因沉默的效果,明确其对花粉发育的影响。【方法】在从白菜花蕾cDNA中克隆BcMF12基因的保守区段的基础上,将白菜花药特异表达启动子BcA9与反义BcMF12基因融合,构建反义表达载体,并经农杆菌介导导入到白菜基因组中,利用抗生素筛选和分子检测筛选出转基因植株。【结果】PCR和Southern检测结果表明,BcA9启动子驱动的反义BcMF12-GUS报告基因已整合到白菜基因组中;Northern杂交显示转基因植株花蕾和花中的BcMF12在mRNA水平的表达明显受到抑制,而且花粉萌发率显著下降。【结论】BcA9调控的反义BcMF12基因明显抑制了BcMF12的表达,从而影响白菜花粉的发育。 相似文献
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为了研究白菜等十字花科植物花粉发育及雄性不育发生的机理,根据编码果胶甲酯酶的白菜雄性不育相关基因BcMF3的cDNA序列设计特异引物,从普通白菜(Brassica campestris ssp.chinensis var.communis,syn.B.rapa ssp.chinensis var.communis)花蕾cDNA中扩增出458 bp的片断,构建BcMF3的反义RNA植物表达栽体,然后转化菜心(B. campestris ssp.chinensis var.parachinensis).研究发现,31.3%菜心转基因植株花粉表现出畸形,而且只有部分花粉具有活力,花粉离体萌发率降低为31.6%,其花药果胶甲酯酶活性降低了12.8%.上述结果表明BcMF3基因与普通白菜和菜心等白菜植物的花粉发育密切相关. 相似文献
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根据普通白菜(Brassica campestris ssp.chinensis var.communis)雄性不育相关基因BcMF4的cDNA序列设计特异引物,从普通白菜花蕾cDNA中扩增出607 bp的片断,然后将该片段连接至双元载体pBI12l中,得到反义RNA植物表达载体并导入农杆菌LBA4404菌株中;通过组织培养途径转化菜心(B.campestris ssp.chinensis var.parachinensis),得到了12株转基因植株,其中5株的43.7%的花粉为缩小空瘪畸形,29.6%的花粉缺乏生活力,而且其花粉离体萌发率降低至24.7%.结果表明,反义RNA技术沉默BcMF4基因导致了菜心转基因植株的部分花粉发育不良,BcMF4基因在普通白菜和菜心等花粉发育中起着重要作用. 相似文献
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根据编码白菜(Brassica rapa L. ssp. chinensis,syn B. campestris ssp. chinensis)果胶甲酯酶的BcMF3 基因cDNA 序列内部第1 343～1 797个碱基之间序列设计引物,从白菜花蕾cDNA中PCR扩增出455 bp的片段,把该片段作为反义基因连接至双元载体pBI12l上,得到了植物表达栽体pBI-B3,并用"三亲杂交"法导入农杆菌LBA4404菌株中,PCR扩增和酶切结果表明所构建的植物表达载体pBI-B3含有BcMF3 反义基因片断,并已导入了根癌农杆菌(Agrobacterinm tumefuciens)中;采用花序浸润法转化拟南芥(Arabidopsis thaliana)后,播种筛选浸润植株的种子获得了5 株卡那霉素抗性植株,PCR 扩增证明BcMF3反义片段成功整合到拟南芥基因组中;转基因拟南芥子代(T2)的抗性分离符合孟德尔分离比例,表明外源基因是单一位点插入. 相似文献
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根据白菜花粉特异的多聚半乳糖醛酸酶基因BcMF2的全长序列设计引物,通过PCR直接扩增的方法从'温州盘菜'芜菁(Brassica campestris L. spp. rapifera cv. Wenzhoupancai)中克隆到了BcMF2的同源基因,命名为BcMF2r.经与白菜BcMF2基因序列比较,DNA全长序列相似性高达90%.Blast搜索结果表明,BcMF2r与拟南芥外切多聚半乳糖醛酸酶在氨基酸水平的的相似性为87%,推测BcMF2r属于多聚半乳糖醛酸酶基因.BcMF2r基因最大开放阅读框为1263 bp,编码420个氨基酸序列,编码的蛋白质分子量为43.8 kDa,等电点为8.108;碱性氨基酸(K, R) 36个;酸性氨基酸(D, E) 32个;疏水氨基酸(A, I, L, F, W, V)152个;极性氨基酸(N, C, Q, S, T, Y)113个.推导的氨基酸序列通过Prosite数据库查询,发现该序列包括3个N-糖基化位点,4个蛋白激酶C磷酸化位点,6个酪蛋白激酶II磷酸化位点,12个N-豆蔻酰化位点, 1个多聚半乳糖醛酸酶活性位点(257RVTCGPGHGLSVGS)等.通过PROFsec预测BcMF2r蛋白质的二级结构,表明该蛋白含7.86% helix,46.90% sheet和45.24% loop.将BcMF2r基因氨基酸序列与数据库中的其他植物PG序列进行同源序列比对,构建系统进化树,发现该基因具有所有PG基因特有的4个保守结构域,并且与花粉中特异表达的PG基因聚为一类,进一步证明了该基因可能是与花粉发育相关的多聚半乳糖醛酸酶基因,在花粉萌发和花粉管伸长中起作用. 相似文献
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The study analyzed the silencing of BcMF12 gene regulated by BcA9 promoter in the transgenic pakchoi and confirmed the effect of antisense BcMF12 gene on the pollen development. A conserved BcMF12 gene fragment was amplified from the cDNA of flower buds in pakchoi （Brassica campestris L. ssp. chinensis, syn. B. rapa L. ssp. chinensis） and was fused to the anther specific BcA9 promoter. The plant antisense expression vector was constructed and then introduced into pakchoi via Agrobacterium-mediated transformation. The transgenic plants were screened by antibiotics and molecular analysis. PCR and Southern blot revealed that the antisense BcMF12-GUS fusion gene regulated by BcA9 promoter was integrated into transgenic plants. Northern blot suggested that the expression of BcMF12 gene was down-regulated significantly. The pollen germination rate of transgenic plants with antisense BcMF12 gene decreased as compared with that of the control plants. The expression of the gene BcMF12 related to the pollen development was inhibited by the antisense BcMF12 driven by BcA9 promoter, which consequently affected the pollen development in pakchoi. 相似文献
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白菜雄性不育相关新基因BcMF1的分离及特征分析 总被引：1，自引：1，他引：0
【目的】筛选与白菜雄性不育性状相关的新基因,为研究植物雄性不育的分子机制提供依据。【方法】利用cDNA-AFLP技术分析白菜核雄性不育两用系‘ZUBajh97-01AB ’的表达差异,在可育株群中扩增出1条特异条带BcMF-A15T17,通过RACE和PCR技术扩增得到该基因的cDNA和DNA全长序列,并用Northern杂交验证了该基因的表达特征。【结果】该基因被命名为BcMF1,它的cDNA全长为1 684 bp,基因全长为1 985 bp,BcMF1基因仅在两用系可育株群的中、大花蕾中特异表达。推测的BcMF1蛋白含有471个氨基酸,与拟南芥未知功能蛋白家族DUF1216成员的相似性较高,预测该蛋白是一个定位在胞外的分泌蛋白。【结论】BcMF1基因是一个与白菜细胞核雄性不育相关的新基因。 相似文献
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利用PCR技术从番茄基因组中扩增了长约1.1kb的E8基因启动子,构建了中间表达载体pCAMBI-AE8;利用RT-PCR方法从拟南芥中扩增了长约1 197bp的2A6基因全长cDNA编码区,将其定向插入pCAMBI-AE8,构建了正义植物表达载体pCAMBIAE8-2A6;同时扩增了长约500bp的cDNA片段,定向插入pCAMBI-AE8,构建了反义植物表达载体pCAMBIAE8-2A6anti.以此为基础,可以进一步研究2A6基因的差异表达对转基因拟南芥角果发育的影响,达到揭示2A6基因功能的目的. 相似文献
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水稻OsCER4基因编码脂肪醛脱羧酶,与拟南芥CER1基因高度同源,是否参与植物表面蜡质合成尚不清楚,为了探讨其功能,扩增了OsCER4基因起始密码子ATG上游约2 kb的序列作为该基因的启动子,以常规技术将启动子和OsCER4基因反义片段克隆到pCAMBIA双元载体1380中,采用农杆菌介导法转化粳稻品种中花11,并进行了转化苗OsCER4蛋白的表达量变化分析.结果表明,成功构建了由OsCER4自身启动子驱动的反义RNA载体,并通过农杆菌介导法成功转入水稻中花11中,多数OsCER4基因反义转化植株为阳性转化植株,后代分离比符合3∶1,反义RNA转化植株在蛋白水平上的表达量下降. 相似文献
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The study analyzed the silencing of BcMF12 gene regulated by BcA9 promoter in the transgenic pakchoi and confirmed the effect of antisense BcMF12 gene on the pollen development. A conserved BcMF12 gene fragment was amplified from the cDNA of flower buds in pakchoi (Brassica campestris L. ssp. chinensis, syn. B. rapa L. ssp. chinensis) and was fused to the anther specific BcA9 promoter. The plant antisense expression vector was constructed and then introduced into pakchoi via Agrobacterium-mediated transformation. The transgenic plants were screened by antibiotics and molecular analysis. PCR and Southern blot revealed that the antisense BcMF12-GUS fusion gene regulated by BcA9 promoter was integrated into transgenic plants. Northern blot suggested that the expression of BcMF12 gene was down-regulated significantly. The pollen germination rate of transgenic plants with antisense BcMF12 gene decreased as compared with that of the control plants. The expression of the gene BcMF12 related to the pollen development was inhibited by the antisense BcMF12 driven by BcA9 promoter, which consequently affected the pollen development in pakchoi. 相似文献
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LIU Le-cheng CAO Jia-shu YU Xiao-lin XIANG Xun FEI Yong-jun 《中国农业科学(英文版)》2006,5(5):339-345
In an effort to provide some information relevant to the molecular mechanism of genic male sterility in plants, BcMF3 gene that encodes a pectin methylesterase was isolated from the fertile B line of Chinese cabbage-pak-choi （Brassica rapa ssp. chinensis, syn. B. campestris ssp. chinensis）. In the present paper, a 455-bp antisense cDNA fragment of BcMF3 was introduced to binary vector pB1121, and then was mobilized into Agrobacterium tumefaciens strain LBA4404. The A. tumefaciens harboring the BcMF3 antisense fragment was transformed to Arabidopsis thaliana by floral dip. Scanning electronic microscopy examination demonstrated that 47.8% of BcMF3 antisense pollen grains exhibited abnormal shape, which might lead to decreased germination of pollens, suggesting that the product of BcMF3 gene plays an important role during microsporogenesis. The evidence on burst of 45.7% of BcMF3 antisense pollen tubes in vitro and a majority of BcMF3 antisense pollens restricted within the stigmatic tissue revealed that BcMF3 is involved in aiding the growth of pollen tubes. The results suggest that BcMF3 acts at both stages of microsporogensis and pollen tube growth. 相似文献
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利用cDNA-AFLP技术分析白菜核雄性不育两用系的表达差异 总被引：23，自引：2，他引：23
利用cDNA AFLP技术 ,对白菜核雄性不育两用系可育株群和不育株群分析显示 :花蕾之间基因表达存在差异 ;莲座期叶片之间、开花期叶片之间和花茎之间基因表达无差异 ;叶片、花茎和花蕾之间基因表达存在差异。对开花期的小蕾、中蕾和大蕾分析显示 :10对引物中 ,1对在可育株中蕾和大蕾中扩增出 1条特异带 ,另 1对扩增出 1条在可育株中蕾中表达丰度高于不育株中蕾的条带 ,其余 8对未发现差异条带。对上述表达丰度有差异的基因进行Northern验证 ,结果与之一致 ,表明cDNA AFLP技术可以用于植物雄性不育基因表达差异的研究。 相似文献
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为研究轮作和有机肥处理对连作土壤的修复作用,以小白菜(Brassica campestris L.ssp.chinensis Makino var.communis Tsen et Lee)连作土壤为对照,比较玉米轮作,玉米秸秆还田,玉米秸秆有机肥等对连作土壤酶活性、微生物活性、活性有机碳含量及小白菜光合速率、根系活力、根系生长的影响。结果表明,轮作和有机肥处理不同程度地增加了连作小白菜光合速率、干物重、根系活力和根系ATPase活性,降低了根系MDA含量,同时提高了连作土壤酸性磷酸酶、脲酶、转化酶活性和微生物对碳源的利用能力,其中长期轮作及施用发酵有机肥的促进作用较大。相关分析表明,土壤酶活性、根系活力、活性有机碳含量、碳源利用AWCD值之间存在极显著正相关关系,说明采用合适的有机肥投入方式可以缓解连作对作物生长造成的抑制。 相似文献