In Silico Cloning and Sequence Analysis of Phospholipase Dα Gene from Peach Fruit

Phospholipase D （PLD, EC 3.1.4.4） plays an important role in adaptive response of postharvest fruit to environment. In this study, a novel cDNA of PLDα was isolated with the strategy of in silico cloning in combination with RT-PCR from peach （Prunus persica L. cv. Jiubao）. The obtained PLDα gene contained a complete open reading frame encoding a 92- kDa protein of 810 amino acid residues, which possessed the characteristic C2 domain and two catalytic HKD motifs. The alignment analysis of the deduced peach PLDa protein with other known PLDα family proteins indicated that peach PLDα was conserved and highly homologous with strawberry PLDα. Semi-quantitative RT-PCR and Northern blot analysis indicated PLDα mRNA in peach fruits could be induced by low temperature. This work provided a scientific basis for further investigating the mechanism of postharvest fruit adaptation to low temperature.     

Molecular Characterization and SNP Markers of the β-purothionin Gene in Einkorn Wheats

Forty-three gene sequences encoding purothionin were characterized from the three species or subspecies of einkorn wheats. These sequences contained 887 bp, among which 92 SNPs including 29 indel loci were detected, giving an average SNP frequency of one SNP per 9.64 bases. According to these sequences, 5 SNP markers were successfully designed, which were used to mine the variations ofpurothionin genes of 102 einkorn wheat accessions. Based on the 5 detected SNP loci, 102 einkorn wheat accessions could be divided into 21 haplotypes, among which 11 haplotypes contained a single sample. Phylogenetic analysis indicated that the purothionin genes from einkorn wheats were more closely related to those from D genome than B genome. Seven out of the 43 gene sequences were assumed to be pseudogenes by the definition of containing in-frame stop codons and small insertions/deletions leading to frameshift. In the remaining 36 amino acid sequences, the 8 Cys and Tyr-13 loci in the mature thionin domain which played important roles in the biological activities were all conserved, whereas there were some varieties occurred in some other important amino acid residues such as Lys and Arg.     

Molecular Identification and Characterization of Cryptosporidium spp. from Mainland China

Three isolates of the genus Cryptosporidium, namely, Guangdong isolate, Anhui isolate and Jiangsu isolate from Mainland China, were identified and characterized genetically utilizing nuclear DNA regions of the small subunit of ribosomal RNA (SSU rRNA) and heat shock protein 70 gene (HSP70) as genetic markers. These two regions were amplified by PCR from DNA extracted from oocysts and amplicons of approximately 290bp and 450bp were produced, respectively. The amplicons were purified, cloned and sequenced. Sequences of 446bp and 290-292bp were obtained for the SSU rRNA and HSP70 regions, respectively. The obtained SSU rRNA and HSP70 sequences representing the three Cryptosporidium isolates were compared with those retrieved from the DNA database. Genetic analyses using either DNA region revealed that members of Cryptosporidium formed two clusters, with C. parvum, C. wariri, C. felis and C. meleagridis clustered together, while C. andersoni, C. muris and C. serpentis belong to the other cluster. Based on SSU rRNA and HSP70 sequences, both Guangdong and Anhui isolates of Cryptosporidium were identified as C. muris of the calf genotype (i.e., C. andersoni), whereas the Jiangsu isolate was identified as C. parvum of the calf genotype. The findings of the present study should have important implications for the diagnosis and control of Cryptosporidium infections in both humans and animals in China.     

Cloning and Identification of S Gene from Chinese Isolate TH—98 of Transmissible Gastroenteritis Virus

Chinese isolate of transmissible gastroenteritis virus(TGEV) was propagated and harvested in swine testicle(ST) cells.Two pairs of primers were designed according to the published sequence with Oligo 4.1 and DNasis softwares.The products of RT-PCR were named Sa and Sb,of 2.3kb and 2.1kb respectively.Sa was inserted in EcoR Iand Kpn I sites after Sb was cloned in Kpm I and Pst I sites of the same pUC18 plasmid,The recombinant designated pUC-S was verified and analyzed by corresponding restriction endonuclease (RE) and nested PCR on the basis of genetic sites of S gene and physical map of pUC18 plasmid,which was identified as S gene from CHinese isolate of TGEV.     

MaCaM在采后香蕉果实温度胁迫及后熟中的作用

【目的】研究香蕉CaM在温度胁迫及香蕉果实后熟过程中的表达模式,了解CaM在增强香蕉果实对温度胁迫的适应性作用,解释CaM参与调控香蕉果实褪绿转黄的机制。【方法】通过比对NCBI数据库中已有物种的CaM氨基酸序列,设计兼并引物。采用热硼酸法,从香蕉果皮中提取总RNA,通过RT-PCR与RACE方法扩增目的基因。利用DNAMAN软件和NCBI网站对CaM的氨基酸序列进行氨基酸比对和同源树分析。利用地高辛探针合成试剂盒（PCR DIG Probe Synthesis Kit）合成特异基因带有DIG标记的探针,使用Northern杂交法对MaCaM在采后香蕉果实温度胁迫及后熟中的表达规律进行分析。钙离子螯合剂EGTA及钙信号恢复处理采用香蕉果皮离体培养,采用真空渗透的方法对香蕉果皮进行试剂处理,利用色差计测定颜色h值。【结果】从香蕉果皮中克隆得到一个CaM,长648 bp,编码138个氨基酸,命名为MaCaM（登录号：HM061077）,序列分析表明,MaCaM包含4个EF-Hand钙离子结合区域,与MaCaM、OsCaM、ZmCaM、AtCaM3、TaCaM1-2等基因同源性极高。Northern杂交结果表明,热激（52℃,3 min）处理香蕉果实0.5 h后,MaCaM表达迅速增强;香蕉果实在冷害温度（7℃）下放置10 d,MaCaM在冷藏的第7-10 d表达逐渐增强,当采后香蕉果实先经热激处理再放入7℃下贮藏,MaCaM表达在第4天和第7天强于7℃处理;乙烯催熟处理诱导香蕉MaCaM表达逐渐增强;30 mmol·L^-1钙离子螯合剂EGTA处理在一定程度上抑制了香蕉果实的后熟,同时也抑制了MaCaM的表达。而在EGTA处理的同时,利用30 mmol·L^-1 CaCl₂进行钙信号恢复处理,能一定程度地恢复香蕉果实的正常后熟,也恢复了MaCaM的表达。【结论】MaCaM能增强香蕉果实对温度胁迫的适应性;MaCaM作为一种调控因子参与了香蕉果实后熟的褪绿转黄过程。     

Isolation and Analysis of α-Gliadin Gene Coding Sequences from Triticum durum

Three coding sequences of gliadins genes, designed as Gli2_Dul, Gli2_Du2 and Gli2_Du3, were isolated from the genomic DNA of Triticum durum accessions CItr5083. Gli2_Dul and Gli2_Du2 contain 945 and 864 bp, encoding the mature proteins with 314 and 287 amino acid residues, respectively. Gli2_Du3 is recognized as a pseudogene due to the stop codon occurring in the coding region. The pseudogenes, commonly occurring in gliadins family, are attributed to the single base change C→T. The amino acid sequences deduced from these gene sequences were characterized with the typical structure of α-gliadin proteins, including the toxic sequences （PSQQQP）. The peptide fraction PF（Y）PP（Q）is thought to be an extra unit of repetitive domain, slightly diverging from the previous report. Six cysteine residues were observed within two unique domains. Phylogenetic analysis showed Gli2_Du2 and Gli2_Du3 were closely related to the genes on chromosome 6A, whereas Gli2_Dul seems to be more homologous with the genes on chromosome 6B.     

Molecular Cloning and Characterization of Pollen Development Related Gene RsMF2 from Raphanus sativus L.

In the paper, the full length cDNA of RsMF2 gene, homologous with the BcMF2 gene encoding pollen-specific polygalacturonase of Chinese cabbage-pak-choi (Brassica campestris L. ssp. chinensis Makino) was cloned from Raphanus sativus L. cv. Yuanbai by PCR, with a pair of primer designed according to the coding sequence of BcMF2. The largest opening reading frame of RsMF2 gene is 1 266 bp in length and encodes a protein of 421 amino acids with a predicted molecular mass of 43.9 kDa. Sequence analysis revealed that it has three potential N-glycosylation sites and one polygalacturonase active position (RVTCGPGHGLSVGS). And the first 32 amino acids of the predicted RsMF2 protein form a N-terminal hydrophobic domain which displays the properties of a signal peptide. The predicted secondary structure composition for the protein has 6.9% helix, 42.0% sheet and 51.1% loop. Four domains which are highly conserved in the whole plant and fungal PGs is present in RsMF2. Phylogenetic analysis showed that RsMF2 falls into the category of clade-C, which includes PGs related to pollen. These results indicate that RsMF2 may act as polygalacturonase related to pollen development.     

玉米catalase-3基因克隆及低温表达研究

应用RT-PCR技术,从玉米自交系承18经14℃冷锻炼处理的芽鞘中扩增出长1491 bp的Cat3全长cDNA。以pGEM-T Easy为载体,克隆了扩增片段,经酶切鉴定和DNA序列测定,确认为玉米Cat3基因。序列分析结果表明,该序列与GenBank中accession number:L05934的序列仅在两个核苷酸位点存在差异。同时采用Northern杂交方法对Cat3在不同低温处理的玉米芽鞘中的表达特性进行了研究,结果显示Cat3在经14℃冷锻炼处理的芽鞘中表达增强,表明该基因受低温的诱导表达,为玉米抗寒基因,对提高玉米的抗寒力起积极作用。     

洋葱γ-谷氨酰转肽酶AcGGT的克隆与鉴定

【目的】葱属植物代谢产生的蒜氨酸具有重要的药学价值,γ-谷氨酰转肽酶是蒜氨酸合成中作为脱谷氨酰化步骤的关键酶。研究洋葱γ-谷氨酰转肽酶基因的功能,揭示γ-谷氨酰转肽酶在洋葱蒜氨酸合成途径中的作用,为体外合成蒜氨酸提供理论依据,为进一步深入研究洋葱蒜氨酸合成机制奠定基础。【方法】以洋葱为材料,依据洋葱RNA-seq数据库设计引物,利用RT-PCR从洋葱中克隆γ-谷氨酰转肽酶基因,并进行生物信息学分析;构建CaMV 35S-AcGGT-GFP载体,利用微粒轰击技术,以金粉-质粒微载体轰击洋葱内表皮细胞,构建带有AcGGT的酿酒酵母表达载体,转化并诱导表达AcGGT,利用γ-谷氨酰转肽酶催化谷氨酰对硝基苯胺生成对硝基苯胺的方法测定转入AcGGT的酿酒酵母总蛋白的谷氨酰转肽酶活性;利用实时荧光定量PCR方法分析该基因在洋葱组织间差异表达模式;利用γ-谷氨酰转肽酶催化谷氨酰对硝基苯胺生成对硝基苯胺的方法测定组织间内源性转肽酶酶活性。【结果】克隆获得AcGGT,长度为1 869 bp;生物学信息学分析显示,洋葱AcGGT编码622个氨基酸,蛋白保守结构域预测显示具有谷氨酰转肽酶结构域,二级结构主要以α-螺旋为主,跨膜区分析推测GGT蛋白具有跨膜区,氨基酸多重比对结果显示植物中的GGT具有一定的保守性,进化分析表明AcGGT与大蒜AsGGT2亲缘关系最接近。CaMV 35S-AcGGT-GFP融合蛋白的荧光信号位于液泡中,表明该基因编码蛋白位于液泡。外源表达AcGGT蛋白的谷氨酰转肽酶活性测定结果显示,转入AcGGT的酵母谷氨酰转肽酶活性显著高于对照,表明AcGGT编码的蛋白具有转肽酶活性。AcGGT组织差异表达结果分析显示,该基因的表达主要在叶鞘,鳞茎和叶鞘次之;不同组织谷氨酰转肽酶活性显示,在叶中活性最高,叶鞘次之。相关性分析显示组织间谷氨酰转肽酶活性与AcGGT表达相关性不显著。【结论】克隆了洋葱AcGGT。洋葱蒜氨酸合成途径中脱谷氨酰化先于S-加氧。AcGGT的表达与洋葱内源性的谷氨酰转肽酶活性相关性不显著,洋葱中可能存在多个谷氨酰转肽酶基因。     

‘京白梨’果实后熟软化与糖、淀粉代谢及其基因表达的关系

【目的】探讨糖和淀粉代谢及其基因表达与京白梨果实软化的关系,为果实贮藏保鲜技术提供依据。【方法】以‘京白梨’果实为试材,经低温和1-MCP处理,测定果实后熟软化过程中硬度、呼吸速率、可溶性糖和淀粉含量及相关酶活性,并对其关键酶基因(AM、SPS、SS和AI)进行实时荧光定量PCR分析。【结果】采后‘京白梨’果实淀粉快速降解,与硬度下降呈极显著正相关关系,低温和1-MCP处理极显著抑制了淀粉含量的下降,降低了其与硬度间的相关水平。同时,淀粉酶(AM)活性快速增加,与硬度和淀粉含量变化极显著相关,且AM的表达量也迅速积累,淀粉酶活性和AM表达量的增加均显著受到低温和1-MCP处理的抑制。常温下,可溶性糖中唯有葡萄糖含量显著下降,果糖和蔗糖含量则有所增加,低温和1-MCP处理则显著抑制了葡萄糖含量的下降以及果糖和葡萄糖含量的增加。蔗糖代谢酶中仅酸性转化酶(AI)活性与果实软化显著相关,其活性和基因表达量的增加时期主要表现在呼吸跃变后期,滞后于AM。蔗糖磷酸合成酶(SPS)和蔗糖合成酶(SS)活性与硬度变化相关性不显著,但随果实软化均表现较高活性和基因表达水平,与蔗糖和果糖间相关性显著,受到低温和1-MCP处理的显著调节。【结论】淀粉降解与‘京白梨’果实软化的关系较为密切,AM是果实软化初期的重要酶;蔗糖代谢参与了‘京白梨’果实后熟软化,AI主要作用于果实后熟软化的后期阶段,SPS和SS能通过调控可溶性糖分的组成和含量来参与果实软化的生理过程。     

低氮胁迫下香蕉硝酸盐转运蛋白MaNRT2基因的克隆与表达分析

氮素是植物生长发育所必需的大量元素,也是限制植物产量的首要因素,硝酸盐转运蛋白NRT是植物吸收和利用氮素的重要蛋白。笔者以香蕉为实验材料,通过RNA-Seq测序,筛选得到一个显著差异表达的高亲和硝酸盐转运蛋白基因NRT2;通过PCR克隆获得1 509 bp的c DNA序列,生物信息学预测其编码502氨基酸,含有MFS结构域,属于NRT2基因家族,命名为MaNRT2;RNA-Seq和qRT-PCR的结果显示,MaNRT2基因的表达具有显著的组织特异性,在根中远高于叶片;低氮胁迫处理后,MaNRT2在叶片中表达量上升,在根中表达量反而下降,表明MaNRT2与香蕉氮元素的吸收转运密切相关。     

橡胶树Lhcb2基因的克隆与表达特性分析

基于从基因组中获得的基因序列,应用RT-PCR技术从橡胶树叶片中分离得到1条849bp的cDNA;该cDNA包含1个798bp的ORF,编码265个氨基酸,理论分子量为2866kD,等电点为542,属于叶绿体定位的类囊体膜蛋白;蛋白含有3个跨膜螺旋,2个C端螺旋,1个保守的捕光叶绿素a/b结合蛋白结构域,1个三聚化基序,以及多个类胡萝卜素和叶绿素结合位点,根据进化关系可归为LHCB2亚家族;蛋白与蓖麻、野草莓、可可、葡萄和棉花中同源蛋白的一致性均接近95%,在进化上显示出高度的保守性,将基因命名为HbLhcb21。表达分析显示,HbLhcb21倾向于在叶片中表达,且其转录水平随着叶片的成熟而逐渐增加,但随着叶片的衰老而明显下调。