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快速碱化因子是近年来新发现的一种植物多肽类信号分子。根据已知的普通白菜两用系中可育株系特异表达的快速碱化因子基因BcMF14序列,在其编码框两侧设计引物,从紫菜薹中克隆出该类信号分子基因,命名为BcMF14p(登陆号:EF523517),全长273 bp,不含内含子,编码框包含222个核苷酸,编码73个氨基酸。NCBI在线同源比对结果表明该基因属于快速碱化因子家族,与花椰菜、拟南芥等的快速碱化因子核苷酸序列相似性达到86%以上。对该信号分子的表达进行RT-PCR分析,结果显示BcMF14p只在紫菜薹花蕾、花、角果等生殖器管中表达,在茎、叶等营养器官中不表达,说明BcMF14p可能参与紫菜薹生殖发育过程中的信号传导。 相似文献
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山药PAL基因全长cDNA序列的克隆、表达与分析 总被引：2，自引：0，他引：2
为了阐明苯丙氨酸解氨酶(PAL,EC4.3.1.5)基因在山药地下块茎酚类物质积累过程中的分子机理,本研究运用普通PCR、RACE和TAIL-PCR技术从大薯(Dioscorea alataL.)’Zixiao’地下块茎中克隆到同一个PAL基因的3个片段,即长度分别为1 145bp、1 151bp和424bp的中间片段、3’端和5’端的cDNA序列;将3段cDNA序列拼接后获得大小为2 376bp的PAL基因全长cDNA序列,该序列含有一个1986bp的最大开放阅读框(ORF),一个28bp的5’端非翻译区,一个362bp含25 nt的Poly(A+)尾的3’端非翻译区,最大ORF可推测编码一个包括PAL酶活性中心的特征序列(GTITASGDLVPLSYIA)在内的661个氨基酸的多肽,分子量为71.974kDa,等电点6.310;大薯地下块茎的PAL基因分别在核苷酸与氨基酸水平上和GenBank中所选已知其他物种的同源性为73%~77%和76%~82%,说明PAL基因在系统进化上相对保守;RT-PCR分析表明该基因仅在地下块茎中表达,而且表达丰度在收获前逐渐降低。 相似文献
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玉米耐旱自交系在干旱条件下的mRNA差异表达 总被引：3，自引：3，他引：3
用16%PEG-6000对玉米耐旱自交系“81565”进行模拟干旱处理,用DD-PCR技术研究干旱与正常浇水对照的mRNA差异表达,发现3个干旱条件下差异表达的特异片段MD1、MD2和MD3。MD1和MD2在干旱条件下减量表达,MD3为干旱诱导表达。序列分析和同源性比对表明,MD1与玉米叶绿体基因组中编码参与RNA转录本Ⅱ型内含子剪切的成熟酶的matK基因有93%的相似性,MD2与极端耐旱的Sporobolus stapfianus的丝氨酸/苏氨酸2C型蛋白磷酸酶基因PP2C的相似性达99%,MD3与属天冬氨酸特异性半胱氨酸蛋白酶类的水稻metacaspase基因有99%的相似性。根据各自序列相似同源基因的功能推测,MD1、MD2和MD3三个片段可能与“81565”的耐旱机制有关。 相似文献
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诸葛菜OvCYP86MF基因的克隆及其特性分析 总被引：2，自引：1，他引：1
为进一步阐明细胞色素P450基因CYP86MF在诸葛菜发育中的分子机理,本文根据已知同源基因保守序列设计特异引物,利用RT-PCR和RACE技术从诸葛菜中获得一个细胞色素P450基因(OvCYP86MF)全长cDNA序列,该序列全长为1876bp,含有1605bp的完整开放阅读框,可编码534个氨基酸,分子量和等电点分别为61.4kDa和6.90,具有细胞色素P450蛋白的典型特征,即保守结构域FNAGPRLCIG;原核表达显示该基因的融合蛋白在体外可以诱导表达;DANSTAR和Clustal W软件分析表明该基因的全长cDNA序列及其编码氨基酸序列与十字花科物种拟南芥相似性很高,达到80%以上,亲缘关系最近;与CYP86C亚家族成员在氨基酸水平上的相似性均高于50%,因此推断该基因属于CYP86C这个亚家族。Northern杂交分析表明该基因在花蕾中特异表达。 相似文献
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根据甘蓝型油菜多聚半乳糖醛酸酶抑制蛋白(PGIP)基因PGIP3的全长序列设计引物,从普通白菜核隐性不育两用系‘Bajh97-01A/B'可育株中成功克隆了多聚半乳糖醛酸酶抑制蛋白基因BcPGIP,对其DNA和cDNA全长序列进行分析,结果表明,该基因包含2个外显子和1个内含子,最大开放阅读框为996bp,编码331个氨基酸,共有10个LRR(Leucine-rich repeat,高氨酸富集)重复序列。将BcPGIP与其他植物的49个PGIPs蛋白进行同源序列比对后,发现PGIP蛋白特征序列非常保守。由重建的进化树可知,该基因与十字花科芸薹属的甘蓝型油菜的同源性最高。通过实时荧光定量PCR分析发现,BcPGIP基因可能与花粉的发育相关。 相似文献
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N+离子注入陆地棉花粉对胚珠DNA及M1代cDNA表达的影响 总被引：1，自引：1，他引：0
通过对胚珠DNA的SSR标记分析,在DNA水平上检测氮离子束诱变后胚珠的多态性变化,结果表明,离子注入陆地棉花粉后再给雌蕊授粉,会对胚珠的DNA多态性产生影响,表明在一定程度上改变了花粉中精细胞的DNA序列;利用抑制性消减杂交(SSH)分离N+离子诱变花粉和对照正常花粉分别给雌蕊授粉后产生的M1代植株叶片间表达有差异的cDNA片段,建立差异表达cDNA文库。已测序的有50个缩减cDNA克隆,根据BLAST网络服务检索查询EMBLGen、 Bank、DDBJ和PDB的核苷酸序列数据库以及蛋白质氨基酸序列数据库,进行序列联配,结果发现52%序列在数据库中都有同源的棉属来源的EST。38个EST与其他物种已知基因部分区域的同源性为56%-100%,占总EST的76%;5条EST序列能在数据库中检索到同源性序列,但其功能（占10%）尚不清楚;9个EST能在数据库中发现为推测蛋白,占18%;4个EST在GenBank中没有查到对应的同源序列,占8%。 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BcMF9的全长序列设计引物，通过PCR直接扩增的方法从结球甘蓝（Brassica. oleracea var. capitata）和芥蓝（B. oleracea var. alboglabra）中克隆得到BcMF9的同源基因BoMF9（BoMF9c和BoMF9l）。生物信息学分析和系统树构建发现BoMF9c和BoMF9l具有花粉特异表达的多聚半乳糖醛酸酶基因的序列特征。不同组织的表达特征分析发现它们分别在结球甘蓝和芥蓝的花蕾中特异表达。推测BoMF9c和BoMF9l可能在花粉萌发和花粉管伸长中起作用。 相似文献
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拟南芥(Arapidopsis)同源异型基因AP2对花分生组织和花器官的发育具有重要的表达调控作用。根据AP2基因信息利用同源序列法分离了油菜(Brassicarapa)的AP2基因(BAP2,GenBank登陆号:AF941097),并且比较了正常油菜与无花瓣油菜BAP2基因序列的差异。研究结果表明,BAP2基因的DNA序列长度为2138bp,包含9个内含子,外显子区与AP2基因的同源性达90%以上;推导的氨基酸序列为433aa,具有完整的核定位信号区和高度保守的AP2结构域,推测与AP2基因具有相似的功能。正常油菜和无花瓣油菜的BAP2基因序列仅有2处碱基存在差异,这2处碱基均位于内含子区,推导的氨基酸序列则完全一致,因此初步认为白菜型油菜花瓣缺失突变与BAP2基因无关。 相似文献
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谷子脂转移蛋白cDNA的克隆及特性研究 总被引：3，自引：0，他引：3
摘要：首次报道了从谷子(Stetaria italica )未成熟种子cDNA文库中克隆到1个与其它物种脂转移蛋白（LTP）具有较高同源性的脂转移蛋白基因Siltp。将核苷酸序列和推断的氨基酸序列在GenBank中进行比较，发现Siltp与大麦脂转移蛋白LTP7a2b和LTPcw-19,玉米脂转移蛋白，欧洲油菜的脂转移蛋白基因的核苷酸序列的同源性分别为72%,70%,60%,54%；氨基酸序列的同源性分别为79%,73%，65%，57%。序列分析表明，编码区含363个碱基 ，编码121个氨基酸，其中包括26个氨基酸的信号肽序列，分子量为9.3 kD。基因组Southern杂交结果表明，Siltp在基因组中以单拷贝存在。Northern杂交研究Siltp的时空表达特异性，发现Siltp仅在茎、叶、穗中表达。利用软件PAUP对17种植物的LTP进行进化分析，从进化系统树可以看出LTP有4个分支，其中，小麦发生分化较早，其它几种单子叶植物如玉米、谷子、大麦和水稻分化较晚，说明发生了早期的基因复制事件。在整个系统图中，双子叶比较分散，说明LTP基因的进化是个复杂的过程；禾本科作物的脂转移蛋白基因是由一个原始基因分化而产生的不同分支，Siltp与大麦LTP亲缘关系最近。 相似文献
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Jégou S Douliez JP Mollé D Boivin P Marion D 《Journal of agricultural and food chemistry》2001,49(10):4942-4949
The influence of malting and brewing processes on the chemical and structural modifications occurring on LTP1 was investigated by mass spectrometry and circular dichroism. Proteins were first purified from malt, and samples were collected at various steps of beer processing performed on two barley cultivars. The levels of LTP1 found in malt were not significantly different from the amounts in barley seed. However, in malt, both LTP1b, a post-translational form of LTP1, and a third isoform named LTP1c were isolated. Moreover, both of these proteins were found to be heterogeneously glycated but still exhibited an alpha-helix structure. Both glycated LTP1 and LTP1b were recovered during mashing. It was also shown that glycated LTP1 was unfolded during heat treatment of wort boiling, which is in agreement with the denatured form previously isolated from beer. 相似文献
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It was previously shown that ungerminated barley contains inhibitors that suppress the activities of green malt cysteine proteinases. This paper reports the purification and partial characterization of a second barley cysteine endoproteinase inhibitor, a protein called lipid transfer protein 2 (LTP2). The chromatographically purified inhibitor had a molecular mass of 7112. The amino acid composition and sequence data of the purified inhibitor indicated that it was a protein whose gene, but not the protein itself, was isolated earlier from barley aleurone tissue. The purified protein inhibited the activities of electrophoretically separated green malt cysteine proteinases but not the activities of the serine- or metalloproteinases. The purified LTP2 inhibited the same proteases as the LTP1 that was characterized previously but was present in the mature seed in much smaller amounts. Neither LTP1 nor LTP2 has been proven to transport lipids in vivo, and it seems possible that both serve to keep cysteine endoproteinases that are synthesized during barley seed development inactive until the plant needs them. The small amount of LTP2 in the seed made it impossible to determine whether it, like LTP1, is involved in beer foam formation. Because of its proteinase-inhibiting ability and its resistance to heat inactivation, some of the LTP2 may persist in beer. 相似文献
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Van Nierop SN Evans DE Axcell BC Cantrell IC Rautenbach M 《Journal of agricultural and food chemistry》2004,52(10):3120-3129
Beer consumers demand satisfactory and consistent foam stability; thus, it is a high priority for brewers. Beer foam is stabilized by the interaction between certain beer proteins, including lipid transfer protein 1 (LTP1), and isomerized hop alpha-acids, but destabilized by lipids. In this study it was shown that the wort boiling temperature during the brewing process was critical in determining the final beer LTP1 content and conformation. LTP1 levels during brewing were measured by an LTP1 ELISA, using antinative barley LTP1 polyclonal antibodies. It was observed that the higher wort boiling temperatures ( approximately 102 degrees C), resulting from low altitude at sea level, reduced the final beer LTP1 level to 2-3 microg/mL, whereas the lower wort boiling temperatures ( approximately 96 degrees C), resulting from higher altitudes (1800 m), produced LTP1 levels between 17 and 35 microg/mL. Low levels of LTP1 in combination with elevated levels of free fatty acids (FFA) resulted in poor foam stability, whereas beer produced with low levels of LTP1 and FFA had satisfactory foam stability. Previous studies indicated the need for LTP1 denaturing to improve its foam stabilizing properties. However, the results presented here show that LTP1 denaturation reduces its ability to act as a binding protein for foam-damaging FFA. These investigations suggest that wort boiling temperature is an important factor in determining the level and conformation of LTP1, thereby favoring satisfactory beer foam stability. 相似文献
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Douliez JP Jégou S Pato C Larré C Mollé D Marion D 《Journal of agricultural and food chemistry》2001,49(4):1805-1808
Recently, this laboratory has isolated from barley and beer extract an isoform of lipid transfer protein (LTP1), which was not fully sequenced (Jégou, S.; Douliez, J. P.; Mollé, D.; Boivin, P.; Marion, D. J. Agric. Food Chem. 2000, 48, 5023--5029). It was named LTP1b and exhibited a molecular weight 294 Da higher than that of the known LTP1. This paper reports the finding of an LTP1 isoform in wheat that also exhibits an excess of 294 Da compared to the native protein. Amino acid sequencing, reduction and alkylation, and mass spectrometry showed that this new LTP1b possesses the same N-terminal sequence as the native LTP1, suggesting that the difference resides in the binding of an adduct which has a molecular weight of 294 Da. The aim of the present paper is to highlight various biophysical techniques that afford the identification of such an isoform-like LTP1 and to correlate this finding with other isoforms of LTP1 that were isolated from other plants but not fully sequenced. 相似文献
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供试菌株Alcaligenes faecalis A15,Enterobacter cloacae EnSs.E·cl-oacae E26,Klebsiella planticola DWUL2.K·oxytoca NG13和Pseudomonassacharophila均能还原2,3,5-氯化三苯基四氮唑(TTC),说明它们含有吸氢酶。以Rhizobium japonicum的hup基因为探针进行杂交的结果表明。除E·oloacae E26和K·oxytoca NG13外,其它菌株的DNA与hup探针具有同源性。A·faecalis A15 hup基因位于染色体上‘E·cloacae EnSs则位于较大的一个质粒上。A·faecalis A15和E·Cloacae EnSs的nif基因与hup基因位于同一复制子上。hup基因不具必然的保守性,因此异源hup基因探针不一定都适宜于探测hup基因。 相似文献
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Capocchi A Fontanini D Muccilli V Cunsolo V Saviozzi F Saletti R Lorenzi R Foti S Galleschi L 《Journal of agricultural and food chemistry》2005,53(20):7976-7984
Isoforms of nonspecific lipid-transfer protein 1 (nsLTP1) and nonspecific lipid-transfer protein 2 (nsLTP2) were investigated in bran tissues isolated from caryopses of two cereal crops quite relevant for the Italian market, the cultivar Centauro of soft wheat (Triticum aestivum) and Italian emmer or farro (Triticum dicoccon Schrank). By sequential separation of the bran extracts on cation-exchange and gel filtration chromatographies, fractions containing only proteins belonging to the nsLTP1 and nsLTP2 classes were obtained. The proteins were roughly identified by SDS-PAGE and by immunoreactions in Western blotting experiments. By MALDI-MS and RP-HPLC/ESI-MS analyses we were able to show the presence of several LTP1 and LTP2 isoforms in the investigated species. Bioinformatic searches based on the determined Mr indicated that (i) two nsLTP1s already identified in T. aestivum have Mr and number of Cys residues identical to that of a 9.6 kDa protein present both in soft wheat cv. Centauro and in farro; (ii) two isoforms of nsLTP2 detected in T. aestivum have the same Mr and number of Cys residues of two 7 kDa proteins found in Centauro; and (iii) a nsLTP1 detected in Ambrosia artemisiifolia has Mr and number of Cys residues coincident to that of a 9.9 kDa protein found both in soft wheat cv. Centauro and in farro. 相似文献
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Stability of barley and malt lipid transfer protein 1 (LTP1) toward heating and reducing agents: relationships with the brewing process 总被引：1，自引：0，他引：1
Perrocheau L Bakan B Boivin P Marion D 《Journal of agricultural and food chemistry》2006,54(8):3108-3113
Barley lipid transfer protein (LTP1) is a heat-stable and protease-resistant albumin that concentrates in beer, where it participates in the formation and stability of beer foam. Whereas the barley LTP1 does not display any foaming properties, the corresponding beer protein is surface-active. Such an improvement is related to glycation by Maillard reactions on malting, acylation on mashing, and structural unfolding on brewing. The structural stability of purified barley and glycated malt LTP1 toward heating has been analyzed. Whatever the modification, lipid adduction or glycation, barley LTP1s are highly stable proteins that resisted temperatures up to 100 degrees C. Unfolding of LTP1 occurred only when heating was conducted in the presence of a reducing agent. In the presence of sodium sulfite, the lipid-adducted barley and malt LTP1 displayed higher heat stability than the nonadducted protein. Glycation had no or weak effect on heat-induced unfolding. Finally, it was shown that unfolding occurred on wort boiling before fermentation and that the reducing conditions are provided by malt extract. 相似文献
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Jégou S Douliez JP Mollé D Boivin P Marion D 《Journal of agricultural and food chemistry》2000,48(10):5023-5029
We report on the purification of lipid transfer proteins (LTP) from barley seeds and beer with the aim of investigating the chemical modifications that occur during the brewing process. In seeds, the well-known LTP of 9 kDa (LTP1) has been found together with a second form named LTPb that displays comparable amino acid composition but was not fully sequenced. These two forms have been recovered in beer with marked chemical modifications including disulfide bond reduction and rearrangement and especially glycation by Maillard reaction. The glycation is heterogeneous with variable amounts of hexose units bound to LTPs. Circular dichroism shows that glycated LTP1 having all their disulfide bridges reduced are totally unfolded. These results provide a first basis for understanding how barley LTPs become foam-promoting agents during the malting and brewing process. 相似文献