油菜角果开裂相关基因的预测及其功能的初步鉴定
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摘要
油菜角果开裂给油菜的生产带来了许多不利的影响,彻底弄清角果开裂过程及其潜在的遗传调节机制能为油菜抗裂角品种的培育及挑选提供新的途径。前期对模式植物拟南芥的研究,鉴定出几个转录调节因子通过调节裂区不同细胞形态的分化形成参与了拟南芥角果的开裂过程。这几个转录因子分别是:FRUITFULL(FUL)、SHATTERPROOF1(SHP1)、SHATTERPROOF2(SHP2)、INDEHISCENT(IND)、ALCATRAZ(ALC)。
为了寻找油菜中以上几个基因的同源基因,本文运用BLAST手段在油菜cDNA库中搜寻到与这几个基因相似性比较高的一些EST序列和cDNA序列。通过拼接、克隆以及RACE方法,获得了几条与FUL、SHP1、SHP2、IND和ALC基因序列同源性比较高的cDNA序列,一个为已知基因BnSHP1,其它的暂命名为甘蓝型油菜的候选基因BnFUL-a、BnFUL-b、BnFUL-c、BnFUL-d、BnSHP2、BnIND和BnALC。
RT-PCR结果显示,BnFUL-b、BnALC、BnSHP2和BnSHP1基因在花和角果中都有显著的表达,此外,BnFUL-b、BnALC、BnSHP2还在其它部位有表达。Southern杂交技术鉴定了BnSHP2基因在甘蓝型油菜基因组中至少存在两个拷贝。
为了鉴定这几个预测基因的功能,本文构建了BnFUL-b基因的过量表达载体和BnALC基因的抑制表达载体,然后通过农杆菌介导转化甘蓝型油菜,通过PCR鉴定了转基因阳性植株。
将BnALC、BnFUL-b、BnIND基因去除了终止密码的ORF片段构建到携带sGFP(S65T)荧光蛋白基因的瞬时表达载体上,通过基因枪轰击洋葱表皮细胞后,在荧光显微镜下观察洋葱表皮细胞发现细胞核内有强烈的荧光产生,表明这几个基因应该定位在细胞核内。
Fruit dehiscence of oilseed rape bring many disadvantages for breeding, therefore,to ravel the dehiscence process and genetic mechanisms underlying the regulation of fruit dehiscence in oilseed rape could suggest new approach to produce new dehiscence tolerant varieties of oilseed rape.Recent studies on Arabidopsis have identified several transcriptional factors involved in fruit dehiscence by regulating the proper differentiation of cell types in dehiscence zone.These transcriptional factors are FRUITFULL(FUL),SHATTERPROOF1(SHP1),SHATTERPROOF2(SHP2), INDEHISCENT(IND),ALCATRAZ(ALC).
To obtain these genes list above in oilseed rape,BLAST procedure was carried out in similarity search in oilseed rape database using FUL,SHP1,SHP2,IND and ALC sequeces as queries respectively,thus some ESTs and cDNAs sequences sharing hingh similarity to queries respectively were obtained.BnSHP1 has been reported and the other candidate genes in Brassica napus containing BnFUL-a、BnFUL-b、BnFUL-c、BnFUL-d、BnSHP2、BnIND and BnALC were finally deduced by sequence assembly,subsequently cloning and RACE method.
The results of RT-PCR showed that BnFUL-b,BnALC,BnSHP1 and BnSHP2 were all expressed in flower and fruit,moreover,BnFUL-b,BnALC and BnSHP2 were also expressed in other vegetative tissues.The southern bloting result showed that there are two duplication of BnSHP2 at least in genome of Brassica napus.
The recombinant plasmid for overexpression of BnFUL-b and the recombinant plasmid for suppressed expression of BnALC were successfully transformed into Brassica napus by Agrobacterium Tumefaciens respectively for further identifying their functions.
The ORF sequences without termination codons of BnFUL-b,BnIND and BnALC were inserted in the position between 35S promoter and sGFP(S65T)gene of the sGFP(S65T)vector respectively,so three transient-expression vectors were constructed and subsequently transferred into onion epidermal cell with particle bombardment.The expressiont of sGFP(S65T)were mainly restricted into the nucleus observed under fluorescence microscope,suggesting that BnFUL-b,BnIND and BnALC proteins were mainly located in the cell nucleus.
为了寻找油菜中以上几个基因的同源基因,本文运用BLAST手段在油菜cDNA库中搜寻到与这几个基因相似性比较高的一些EST序列和cDNA序列。通过拼接、克隆以及RACE方法,获得了几条与FUL、SHP1、SHP2、IND和ALC基因序列同源性比较高的cDNA序列,一个为已知基因BnSHP1,其它的暂命名为甘蓝型油菜的候选基因BnFUL-a、BnFUL-b、BnFUL-c、BnFUL-d、BnSHP2、BnIND和BnALC。
RT-PCR结果显示,BnFUL-b、BnALC、BnSHP2和BnSHP1基因在花和角果中都有显著的表达,此外,BnFUL-b、BnALC、BnSHP2还在其它部位有表达。Southern杂交技术鉴定了BnSHP2基因在甘蓝型油菜基因组中至少存在两个拷贝。
为了鉴定这几个预测基因的功能,本文构建了BnFUL-b基因的过量表达载体和BnALC基因的抑制表达载体,然后通过农杆菌介导转化甘蓝型油菜,通过PCR鉴定了转基因阳性植株。
将BnALC、BnFUL-b、BnIND基因去除了终止密码的ORF片段构建到携带sGFP(S65T)荧光蛋白基因的瞬时表达载体上,通过基因枪轰击洋葱表皮细胞后,在荧光显微镜下观察洋葱表皮细胞发现细胞核内有强烈的荧光产生,表明这几个基因应该定位在细胞核内。
Fruit dehiscence of oilseed rape bring many disadvantages for breeding, therefore,to ravel the dehiscence process and genetic mechanisms underlying the regulation of fruit dehiscence in oilseed rape could suggest new approach to produce new dehiscence tolerant varieties of oilseed rape.Recent studies on Arabidopsis have identified several transcriptional factors involved in fruit dehiscence by regulating the proper differentiation of cell types in dehiscence zone.These transcriptional factors are FRUITFULL(FUL),SHATTERPROOF1(SHP1),SHATTERPROOF2(SHP2), INDEHISCENT(IND),ALCATRAZ(ALC).
To obtain these genes list above in oilseed rape,BLAST procedure was carried out in similarity search in oilseed rape database using FUL,SHP1,SHP2,IND and ALC sequeces as queries respectively,thus some ESTs and cDNAs sequences sharing hingh similarity to queries respectively were obtained.BnSHP1 has been reported and the other candidate genes in Brassica napus containing BnFUL-a、BnFUL-b、BnFUL-c、BnFUL-d、BnSHP2、BnIND and BnALC were finally deduced by sequence assembly,subsequently cloning and RACE method.
The results of RT-PCR showed that BnFUL-b,BnALC,BnSHP1 and BnSHP2 were all expressed in flower and fruit,moreover,BnFUL-b,BnALC and BnSHP2 were also expressed in other vegetative tissues.The southern bloting result showed that there are two duplication of BnSHP2 at least in genome of Brassica napus.
The recombinant plasmid for overexpression of BnFUL-b and the recombinant plasmid for suppressed expression of BnALC were successfully transformed into Brassica napus by Agrobacterium Tumefaciens respectively for further identifying their functions.
The ORF sequences without termination codons of BnFUL-b,BnIND and BnALC were inserted in the position between 35S promoter and sGFP(S65T)gene of the sGFP(S65T)vector respectively,so three transient-expression vectors were constructed and subsequently transferred into onion epidermal cell with particle bombardment.The expressiont of sGFP(S65T)were mainly restricted into the nucleus observed under fluorescence microscope,suggesting that BnFUL-b,BnIND and BnALC proteins were mainly located in the cell nucleus.
引文
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[3]Pekrun C,Lutman P J W,Baeumer K.Germination behaviour of dormant oilseed rape seeds in relation to temperature.Weed Research 1997;37(6):419-431.
[4]Pekrun C,Lutman P J W,Baeumer K.Induction of secondary dormancy in rape seeds (Brassica napus L.)by prolonged imbibition under conditions of water stress or oxygen deficiency in darkness.European Journal of Agronomy 1997;6(3-4):245-255.
[5]Pekrun C,Hewitt J D J,Lutman P J W.Cultural control of volunteer oilseed rape(Brassica napus).The Journal of Agricultural Science 1998;130.
[6]Lopez G F,Lutman P J W.Effect of Environmental Conditions on the Dormancy and Germination of Volunteer Oilseed Rape Seed(Brassica napus).Weed Science 1998;46(4):419-423.
[7]Ostergaard L,Kempin S A,Bies D,Klee H J,Yanofsky M F.Pod shatter-resistant Brassica fruit produced by ectopic expression of the FRUITFULL gene.Plant Biotechnol J 2006;4(1):45-51.
[8]Morgan C L,Bruce D M,Child R.Genetic variation for pod shatter resistance among lines of oilseed rapadeveloped from synthetic B.napus.Field Crops Reasearch 1998;58:153-165.
[9]Bowman J L,Baum S F,Eshed Y,Putterill J,Alvarez J.Molecular genetics ofgynoecium development in Arabidopsis.Curr Top Dev Biol 1999;45:155-205.
[10]Ferrandiz C,Pelaz S,Yanofsky M F.Control of carpel and fruit development in Arabidopsis.Annu Rev Biochem 1999;68:321-54.
[11]Ferrandiz C.Regulation of fruit dehiscence in Arabidopsis.J Exp Bot 2002;53(377):2031-8.
[12]Smyth D,Bowman J,Meyerowitz E.Early Flower Development in Arabidopsis.The Plant Cell 1990;2(8):755-767.
[13]Spence J,Vercher Y,Gates P,Harris N.Pod shatter' in Arabidopsis thaliana,Brassica napus and B.j uncea.Journal of Microscopy 1996;181:195-203.
[14]Meakin P,Roberts J.Dehiscence of fruit in oilseed rape.Ⅱ.The role of cell wall degrading enzymes.Journal of Experimental Botany 1990;41:1003-1011.
[15]Rajani S,Sundaresan V.The Arabidopsis myc/bHLH gene ALCATRAZ enables cell separation in fruit dehiscence.Curr Biol 2001;11(24):1914-22.
[16]Meakin P,Roberts J.Dehiscence of fruit in oilseed rape.Ⅰ.Anatomy of pod dehiscence.Journal of Experimental Botany 1990;41:995-1002.
[17]Liljegren S J.SHATTERPROOF MADS-box genes control seed dispersal in Arabidopsis.Nature 2000;404:766-770.
[18]Sundaresan V,Springer P,Volpe T,Haward S,Jones J D,Dean C,Ma H,Martienssen R.Patterns of gene action in plant development revealed by enhancer trap and gene trap transposable elements.Genes Dev 1995;9(14):1797-810.
[19]Gu Q,Ferrandiz C,Yanofsky M F,Martienssen R.The FRUITFULL MADS-box gene mediates cell differentiation during Arabidopsis fruit development. Development 1998; 125(8): 1509-17.
[20] Mandel M A, Yanofsky M F. The Arabidopsis AGL8 MADS box gene is expressed in inflorescence meristems and is negatively regulated by APETALA1. Plant Cell 1995; 7(11): 1763-71.
[21] Ferrandiz C, Liljegren S J, Yanofsky M F. Negative regulation of the SHATTERPROOF genes by FRUITFULL during Arabidopsis fruit development. Science 2000; 289(5478): 436-8.
[22] Liljegren S, Kempin S, Chen A, Roeder A, Guimil S, Khammungkhune T, Yanofsky M. A bHLH gene, INDEHISCENT1, is required for fruit dehiscence and mediates the fruitfull phenotype. in 11th International conference on Arabidopsis research. 2000. Madison, WI. USA: University of Madison Press.
[23] Liljegren S J, Roeder A H, Kempin S A, Gremski K, Ostergaard L, Guimil S, Reyes D K, Yanofsky M F. Control of fruit patterning in Arabidopsis by INDEHISCENT. Cell 2004; 116(6): 843-53.
[24] Wu H, Mori A, Jiang X, Wang Y, Yang M. The INDEHISCENT protein regulates unequal cell divisions in Arabidopsis fruit. Planta 2006; 224(4): 971-9.
[25] Roeder A, Liljegren S, Eshed Y, Bowman J, Alonso J, Ecker J, Yanofsky M. Enhancer trap lines YJ161 and YJ115 are expressed in the developing fruit. In 12th International conference on Arabidopsis research. 2001. Madison, WI, USA: University of Wisconsin Press.
[26] Patterson S E. Cutting loose. Abscission and dehiscence in Arabidopsis. Plant Physiol 2001; 126(2): 494-500.
[27] Roberts J A, Whitelaw C, Gonzalez-Carranza Z, MeManus M. Cell separation processes in plants: models, mechanisms and manipulation. Annals of Botany 2000; 86: 223-235.
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