马铃薯α-淀粉酶基因的克隆及生物信息学分析(英文)

本研究利用RT-PCR从马铃薯(Solanum tuberosum)茎段总RNA中扩增、克隆了一cDNA分子。该cDNA分子含有一长为1224bp的开放读框,可编码一含407个氨基酸残基的多肽、理论分子量为46.40kD、可能为亲水性的胞外酶。因其氨基酸序列同源于α-淀粉酶,故将该基因命名为amyA1(NCBI收录号:GQ406048.1)。采用半定量RT-PCR方法检测了amyA1基因在马铃薯茎、叶等不同组织中的表达强度,表明在茎组织中的表达丰度略高。利用生物信息学软件分析了amyA1密码子的偏好性,以期为选择适宜的表达系统提供依据;同时对amyA1的理化性质、细胞内定位、保守结构及高级结构进行了预测。基于NCBI数据库中有物种代表性的29种α-淀粉酶基因序列构建了基因进化树。与NCBI收录的马铃薯α-淀粉酶基因(NCBI收录号:M79328.1)的核苷酸及氨基酸序列同源性达98%。第20至第348范围内的氨基酸残基含有与淀粉酶13家族及亚家族相似的催化活性域(PF00128、SM00624),第349至第407范围内的氨基酸残基含有α-淀粉酶C-末端β折叠区域(PF07821)。蛋白质结构预测表明氨基酸残基序列有维持淀粉酶活性的(β/α)8桶状结构以及其它几个功能域结构。所构建的基因进化树表明,2个马铃薯α-淀粉酶基因与木薯、苹果的序列同源性较高,与菜豆的次之,与水稻、大麦和玉米等单子叶植物的序列同源性较低。     

Introduction of sense and antisense cDNA for branching enzyme in the amylose-free potato mutant leads to physico-chemical changes in the starch

One isoform of the branching enzyme (BE; EC 2.4.1.18) of potato (Solarium tuberosum L.) is known and catalyses the formation of α-1,6 bonds in a glucan chain, resulting in the branched starch component amylopectin. Constructs containing the antisense or sense-orientated distal 1.5-kb part of a cDNA for potato BE were used to transform the amylose-free (amf) mutant of potato, the starch of which stains red with iodine. The expression of the endogenous BE gene was inhibited either largely or fully as judged by the decrease or absence of the BE mRNA and protein. This resulted in a low percentage of starch granules with a small blue core and large red outer layer. There was no effect on the amylose content, degree of branching or λ_max of the iodine-stained starch. However, when the physico-chemical properties of the different starch suspensions were assessed, differences were observed, which although small indicated that starch in the transformants was different from that of theamf mutant.     

马铃薯POTHR-1 cDNA的克隆及晚疫病菌和其他非生物因子诱导表达分析

利RACE和重叠延伸相结合的方法,从经晚疫病菌接种诱导的马铃薯水平抗性材料叶片中克隆了一个POTHE 1基因(potato Phytophthora infestans induced hypersensitive response related protein gene)的全长cDNA.序列分析表明,该基因编码225个氨基酸,与烟草harpin诱导蛋白基因hinl有很高的同源性(编码区核苷酸和氨基酸序列分别为83%和81%).Southern杂交结果显示在马铃薯基因组中有2～3个拷贝.对其诱导表达模式研究表明:晚疫病病原菌接种36 h后,该基因表达迅速增加;机械伤害及茉莉酸(JA)处理能够诱导表达;渗透胁迫(NaCl浸泡)能够诱导其微弱表达;但水杨酸(SA)不能诱导表达.该基因可能和病原与寄主互作时寄主产生过敏反应及细胞生理性死亡有关.     

马铃薯POTHR-1 cDNA的克隆及晚疫病菌和其他非生物因子诱导表达分析

利用RACE和重叠延伸相结合的方法,从经晚疫病菌接种诱导的马铃薯水平抗性材料叶片中克隆了一个POTHR-I基因(potato Phytophthora infestans-induced hypersensitive response related protein gene)的全长cDNA。序列分析表明,该基因编码225个氨基酸,与烟草harpin诱导蛋白基因hinI有很高的同源性(编码区核苷酸和氨基酸序列分别为83％和81％)。Southern杂交结果显示在马铃薯基因组中有2、3个拷贝。对其诱导表达模式研究表明：晚疫病病原菌接种36h后,该基因表达迅速增加;机械伤害及茉莉酸(JA)处理能够诱导表达;渗透胁迫(NaCI浸泡)能够诱导其微弱表达;但水杨酸(SA)不能诱导表达。该基因可能和病原与寄主互作时寄主产生过敏反应及细胞生理性死亡有关。     

Molecular cloning and characterization of a cDNA encoding endonuclease from potato (Solanum tuberosum)

A cDNA, StEN1, encoding a potato (Solanum tuberosum) endonuclease was cloned and sequenced. The nucleotide sequence of this clone contains an open reading frame of 906 nucleotides encoding a protein of 302 amino acids, and with a calculated molecular mass of 34.4kDa and a Pi of 5.6. The deduced StEN1 protein contains a putative signal sequence of 25 amino acid residues. The StEN1 encoded protein shows substantial homology to both plant and fungal endonucleases isolated and cloned from other sources. The highest identity (73%) was observed with AgCEL I from celery, Apium graveolens, ZEN1 from Zinnia elegans (69%) and DSA6 from daylily, Hemerocallis (68%). RT-PCR expression analysis demonstrated that the potato StEN1 gene is constitutively expressed in potato, although minor differences in expression level in different tissues were observed.     

Starch branching enzymes belonging to distinct enzyme families are differentially expressed during pea embryo development

cDNA clones for two isoforms of starch branching enzyme (SBEI and SBEII) have been isolated from pea embryos and sequenced. The deduced amino acid sequences of pea SBEI and SBEII are closely related to starch branching enzymes of maize, rice, potato and cassava and a number of glycogen branching enzymes from yeast, mammals and several prokaryotic species. In comparison with SBEI, the deduced amino acid sequence of SBEII lacks a flexible domain at the N-terminus of the mature protein. This domain is also present in maize SBEII and rice SBEIII and resembles one previously reported for pea granule-bound starch synthase II (GBSSII). However, in each case it is missing from the other isoform of SBE from the same species. On the basis of this structural feature (which exists in some isoforms from both monocots and dicots) and other differences in sequence, SBEs from plants may be divided into two distinct enzyme families. There is strong evidence from our own and other work that the amylopectin products of the enzymes from these two families are qualitatively different. Pea SBEI and SBEII are differentially expressed during embryo development. SBEI is relatively highly expressed in young embryos whilst maximum expression of SBEII occurs in older embryos. The differential expression of isoforms which have distinct catalytic properties means that the contribution of each SBE isoform to starch biosynthesis changes during embryo development. Qualitative measurement of amylopectin from developing and maturing embryos confirms that the nature of amylopectin changes during pea embryo development and that this correlates with the differential expression of SBE isoforms.     

Cloning and characterization of a hydroxycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl)transferase induced in response to UV-C and wounding from Capsicum annuum

Hydroxycinnamoyl-CoA : tyramine N-(hydroxycinnamoyl) transferase (THT) is a pivotal enzyme in the synthesis of N-(hydroxycinnamoyl)-amines, which are associated with cell wall fortification in plants. The cDNA encoding THT was cloned from the leaves of UV-C treated Capsicum annuum (hot pepper) using a differential screening strategy. The predicted protein encoded by the THT cDNA is 250 amino acids in length and has a relative molecular mass of 28,221. The protein sequence derived from the cDNA shares 76% and 67% identity with the potato and tobacco THT protein sequences, respectively. The recombinant pepper THT enzyme was purified using a bacterial overexpression system. The purified enzyme has a broad substrate specificity including acyl donors such as cinnamoyl-, sinapoyl-, feruloyl-, caffeoyl-, and 4-coumaroyl-CoA and acceptors such as tyramine and octopamine. In UV-C treated plants, the THT mRNA was strongly induced in leaves, and the elevated level of expression was stable for up to 36 h. THT mRNA also increased in leaves that were detached from the plant but not treated with UV-C. THT expression was measured in different plant tissues, and was constitutive at a similar level in leaf, root, stem, flower and fruit. Induction of THT mRNA was correlated with an increase in THT protein.     

Expression of a cassava granule-bound starch synthase gene in the amylose-free potato only partially restores amylose content

Granule-bound starch synthase I (GBSS I) is responsible for the synthesis of amylose in starch granules. A heterologous cassava GBSS I gene was tested for its ability to restore amylose synthesis in amylose-free (amf) potato mutants. For this purpose, the cassava GBSS I was equipped with different transit peptides. In addition, a hybrid containing the potato transit peptide, the N-terminal 89 amino acids of the mature potato GBSS I, and the C-terminal part of cassava GBSS I was prepared. The transgenic starches were first analysed by iodine staining. Only with the hybrid could full phenotypic complementation of the amf mutation be achieved in 13% of the plants. Most transformants showed partial complementation, but interestingly the size of the blue core was similar in all granules derived from one tuber of a given plant. The amylose content was only partially restored, up to 60% of wild-type values or potato GBSS I-complemented plants; however, the GBSS activity in these granules was similar to that found in wild-type ones. From this, and the observation that the hybrid protein (a partial potato GBSS I look-alike) performs best, it was concluded that potato and cassava GBSS I have different intrinsic properties and that the cassava enzyme is not fully adapted to the potato situation.     

紫色马铃薯类黄酮3，′5′-羟化酶基因（StF3′5′H）的克隆及分析

类黄酮3′,5′羟-化酶（ flavonoid 3′,5′-hydroxylase, F3′5′H）是植物花青素生物合成途径中的一个关键酶,紫色土豆（ Solanum tueb or sum） F3′5′H基因的克隆将为花青素合成调控和花青素代谢工程研究提供优质基因资源。研究采用RACE技术克隆了紫色土豆F3′5′H基因的cDNA全长序列,用生物信息学方法对其核苷酸和蛋白质序列进行了分析,并用半定量PCR 技术分析了F3′5′H基因在不同组织中的表达情况,同时研究了赤霉素和蔗糖处理后F3′5′H基因表达与花青素积累之间的相关性。研究结果表明,克隆的紫色土豆F3′5′H的cDNA全长为1854 bp,包含一个1530 bp的完整ORF,共编码509个氨基酸。生物信息学分析表明,StF3′5′H基因推测编码的氨基酸序列与其它植物的F3′5′H蛋白的相似性很高。 StF3′5′H基因的表达具有组织特异性,在紫色土豆根、茎和叶柄中都有表达,其中在叶柄中表达最强,而在块茎、叶轴和叶片中几乎检测不到StF3′5′H基因的表达。赤霉素和蔗糖能促进紫色土豆StF3′5′H基因的表达,进而促进花青素的积累。     

菠萝蜜低聚肽对db/db小鼠炎症反应、血糖及血脂的影响

目的：研究菠萝蜜低聚肽（JOPs）干预对db/db糖尿病模型小鼠炎症反应、血糖及血脂的影响作用。方法：选择db/db糖尿病小鼠模型,将其随机分为3个JOPs组（0.2、0.4、0.8g/kg·BW）以及糖尿病模型对照组、二甲双胍对照组、乳清蛋白对照组,并选用db/m小鼠作为非糖尿病小鼠空白对照。经过为期6个月的干预,检测小鼠空腹血糖（FPG）、血清胰岛素（INS）、白细胞介素6（IL 6）、白细胞介素8（IL 8）、白细胞介素10（IL 10）和肿瘤坏死因子α（TNF α）、C反应蛋白（CRP）以及脂代谢指标。结果：JOPs可显著降低db/db糖尿病小鼠空腹血糖水平及胰岛素抵抗指数;可使血清IL 6、TNF α、总胆固醇（TC）和甘油三酯（TG）显著降低,并使高密度脂蛋白胆固醇（HDL C）显著升高。结论：JOPs干预可有效降低糖尿病小鼠的血糖水平,改善胰岛素抵抗,同时有效调节炎症反应及血脂代谢。     

Isolation and sequencing of cDNA clones encoding ethylene-induced putative peroxidases from cucumber cotyledons

A cDNA library from ethephon-treated cucumber cotyledons (Cucumis sativus L. cv. Poinsett 76) was constructed. Two cDNA clones encoding putative peroxidases were isolated by means of a synthetic probe based on a partial amino acid sequence of a 33 kDa cationic peroxidase that had been previously shown to be induced by ethylene. DNA sequencing indicates that the two clones were derived from two closely related RNA species that are related to published plant peroxidase sequences. Southern analysis indicates that there are 1–5 copies in a haploid genome of a gene homologous to the cDNA clones. The deduced amino acid sequences are homologous with a tobacco (55% sequence identity), a horseradish (53%), a turnip (45%), and a potato (41%) peroxidase. The cloned sequences do not encode the 33 kDa peroxidase from which the original synthetic probe was been derived, but rather other putative peroxidases. An increase in the level of mRNA is evident by 3 hours after ethephon or ethylene treatment and plateaus by 15 hours.