Characteristics and phylogeny of light-harvesting complex gene encoded proteins from marine red alga Griffithsia japonica

Six genes encoding light-harvesting complex (LHC) protein have been characterized in the multicellular red alga Griffithsia japonica EST analysis. Three of them were full sequences while others were partial sequences with 3'-UTRs. The cleavage sites between signal peptide and mature LHC protein were analyzed on these three full sequences. The sequence characteristics, calculated molecular weights and isoelectric point (pI) values and hydrophobicity of the mature proteins were deduced and analyzed. Comparing the LHC sequences of G. japonica with higher plant, Chlorophyta, chromophytes and other red algae, the high conservation of the chlorophyll (Chl) binding site among chromophytes and red algae were revealed. Phylogenetic analysis on LHC proteins from higher plant, green algae, euglena, brown algae, diatom, cryptomonad, Raphidophyte and red algae reveals that (1) there are two distinct groups of Chl a/b and Chl a/c -binding LHC; (2) Chl a binding proteins of red algae share greater similarities with the Chl a/c-binding proteins of the chromophytes and dinoflagellate than with the Chl a/b - binding proteins of the green algae and higher plants; (3) chromophyte's LHC is supposed to be evolved from red algae LHC.

Differential gene expression in the body wall of the sea cucumber(Apostichopus japonicus)under strong lighting and dark conditions

Sea cucumber, Apostichopus japonicus is very sensitive to light changes. It is important to study the influence of light on the molecular response of A. japonicus. In this study, RNA-seq provided a general overview of the gene expression profiles of the body walls of A. japonicus exposed to strong light（＂light＂）, normal light（＂control＂） and fully dark（＂dark＂） environment. In the comparisons of ＂control＂ vs. ＂dark＂, ＂control＂ vs. ＂light＂ and ＂dark＂ vs.＂light＂, 1 161, 113 and 1 705 differentially expressed genes（DEGs） were identified following the criteria of｜log2 ratio｜≥1 and FDR≤0.001, respectively. Gene ontology analysis showed that ＂cellular process＂ and ＂binding＂enriched the most DEGs in the category of ＂biological process＂ and ＂molecular function＂, while ＂cell＂ and ＂cell part＂ enriched the most DEGs in the category of ＂cellular component＂. And the DEGs were mapped to 214, 41 and229 pathways in the Kyoto Encyclopedia of Genes and Genomes database, and 51, 2 and 57 pathways were significantly enriched, respectively. Light-specific DEGs identified in this study will be important targets for further investigation to establish the biochemical mechanisms involved in the adaption of this sea cucumber to changes in the level of environmental light.

Genetic Analysis and Molecular Mapping of Light-Sensitive Red-Root Mutant in Rice

The light-sensitive red-root mutant, designated as HG1, was newly observed from an indica rice variety, Nankinkodo, when seedlings were grown with roots exposed to natural light. The root color of the mutant began to turn slight-red when the roots were exposed to the light at the intensity of 29 ）Jmol/（m^2·s）, then turned dark-red at the light intensity of 180 pmol/（m^2·s）, suggesting that the root color of the mutant was evidently sensitive to light. Furthermore, genetic analysis showed that the character of light-sensitive red-root of the HG1 mutant was controlled by a single dominant gene, tentatively designated as Lsr. With simple sequence repeat markers, Lsrgene was located between the markers RM252 and RM303 on chromosome 4 with the genetic distances of 9.8 cM and 6.4 cM, respectively. These results could be useful for fine mapping and cloning of Lsrgene in rice.

Studies Shed Light on the Albinism Gene of Rhesus Monkey

A recent study by researchers at the Kunming Institute of Zoology (KIZ) of the Chinese Academy of Sciences (CAS) identifies the albinism gene of rhesus monkeys using the method of molecular technology, and suggests the age of the albinism gene in rhesus monkeys should be roughly 800,000 years. The general albinism

Analysis of the autophagy gene expression profile of pancreatic cancer based on autophagy-related protein microtubule-associated protein 1A/1B-light chain 3

BACKGROUND Pancreatic cancer is a highly invasive malignant tumor. Expression levels of the autophagy-related protein microtubule-associated protein 1 A/1 B-light chain 3(LC3) and perineural invasion(PNI) are closely related to its occurrence and development. Our previous results showed that the high expression of LC3 was positively correlated with PNI in the patients with pancreatic cancer. In this study, we further searched for differential genes involved in autophagy of pancreatic cancer by gene expression profiling and analyzed their biological functions in pancreatic cancer, which provides a theoretical basis for elucidating the pathophysiological mechanism of autophagy in pancreatic cancer and PNI.AIM To identify differentially expressed genes involved in pancreatic cancer autophagy and explore the pathogenesis at the molecular level.METHODS Two sets of gene expression profiles of pancreatic cancer/normal tissue(GSE16515 and GSE15471) were collected from the Gene Expression Omnibus.Significance analysis of microarrays algorithm was used to screen differentially expressed genes related to pancreatic cancer. Gene Ontology(GO) analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway analysis were used to analyze the functional enrichment of the differentially expressed genes. Protein interaction data containing only differentially expressed genes was downloaded from String database and screened. Module mining was carried out by Cytoscape software and ClusterOne plug-in. The interaction relationship between the modules was analyzed and the pivot nodes between the functional modules were determined according to the information of the functional modules and the data of reliable protein interaction network.RESULTS Based on the above two data sets of pancreatic tissue total gene expression, 6098 and 12928 differentially expressed genes were obtained by analysis of genes with higher phenotypic correlation. After extracting the intersection of the two differential gene sets, 4870 genes were determined. GO analysis showed that 14 significant functional items including negative regulation of protein ubiquitination were closely related to autophagy. A total of 986 differentially expressed genes were enriched in these functional items. After eliminating the autophagy related genes of human cancer cells which had been defined, 347 differentially expressed genes were obtained. KEGG pathway analysis showed that the pathways hsa04144 and hsa04020 were related to autophagy. In addition,65 clustering modules were screened after the protein interaction network was constructed based on String database, and module 32 contains the LC3 gene,which interacts with multiple autophagy-related genes. Moreover, ubiquitin C acts as a pivot node in functional modules to connect multiple modules related to pancreatic cancer and autophagy.CONCLUSION Three hundred and forty-seven genes associated with autophagy in human pancreatic cancer were concentrated, and a key gene ubiquitin C which is closely related to the occurrence of PNI was determined, suggesting that LC3 may influence the PNI and prognosis of pancreatic cancer through ubiquitin C.

Isolation and Purification of Carp Mitochondrial DNA and Structural Analysis of Its tRNA^(Cys) Gene and the Light Strand Origin

A new method is presented with which we isolated milochondrial DNA from fresh carp liver usingdifferential centrifugation and DNase treatment that gave high yield of purified product with an easyand economical procedure. Highly distinct bands were displayed in agarose gel electrophoresls ofthe product digested with restrictlon enzymes, which were successfully used in constructingrestriction map and molecular clone of mitochondrial genes. With DNAs thus obtained, we havecloned cysteine tRNA gene (tRNA^(Cys) gene) of carp mitochondria, determined the nucleotide sequenceof it and the light strand origin, and depicted the cloverleaf secondary structure of tDNA^(Cya) and thelight strand origin. Analysis of nucleotide sequences of tRNA^(Cy) genes of 5 vertebrates has revealedunusual features of carp mitochondrial tRNA^(Cy) gene as compared with their cytoplasmic counter-parts, Altogether 36 bases were found in the light strand origin of carp mitochondriaf: 11 pairs in thestem; and 14 bases in the loop. As compared with those of other 11 vertebrate species, the sequenceof the stem is very conservative while both sequence and length of the loop are quite variable. Thestructure of the stem-loop may play an important role in light strand replication.

Identification of Light-Harvesting Chlorophyll a/b-Binding Protein Genes of Zostera marina L.and Their Expression Under Different Environmental Conditions

Photosynthesis includes the collection of light and the transfer of solar energy using light-harvesting chlorophyll a/b-binding(LHC) proteins.In high plants,the LHC gene family includes LHCA and LHCB sub-families,which encode proteins constituting the light-harvesting complex of photosystems I and II.Zostera marina L.is a monocotyledonous angiosperm and inhabits submerged marine environments rather than land environments.We characterized the Lhca and Lhcb gene families of Z.marina from the expressed sequence tags(EST) database.In total,13 unigenes were annotated as Zm Lhc,6 in Lhca family and 7 in Zm Lhcb family.Zm LHCA and Zm LHCB contained the conservative LHC motifs and amino acid residues binding chlorophyll.The average similarity among mature Zm LHCA and Zm LHCB was 48.91% and 48.66%,respectively,which indicated a high degree of divergence within Zm LHChc gene family.The reconstructed phylogenetic tree showed that the tree topology and phylogenetic relationship were similar to those reported in other high plants,suggesting that the Lhc genes were highly conservative and the classification of Zm Lhc genes was consistent with the evolutionary position of Z.marina.Real-time reverse transcription(RT) PCR analysis showed that different members of Zm Lhca and Zm Lhcb responded to a stress in different expression patterns.Salinity,temperature,light intensity and light quality may affect the expression of most Zm Lhca and Zm Lhcb genes.Inorganic carbon concentration and acidity had no obvious effect on Zm Lhca and Zm Lhcb gene expression,except for Zm Lhca6.

人LIGHT基因的克隆及其重组腺病毒载体的构建

目的 克隆人ＬＩＧＨＴ基因，构建其重组腺病毒载体，以研究ＬＩＧＨＴ过表达与腺病毒感染对细胞生长的影响。方法用ＲＴ－ＰＣＲ法从人外周血单个核细胞（ＰＢＭＣ）中克隆人的ＬＩＧＨＴ全长基因。将ＬＩＧＨＴ ｃＤＮＡ克隆到穿棱载体ｐＡｄＴｒａｃｋ－ＣＭＶ－ＬＩＧＨＴ重组质粒中，经酶切线性化后，将重组质粒ｐＡｄＴｒａｃｋ－ＣＭＶ－ＬＩＧＨＴ和骨架质粒ｐＡｄＥａｓｙ－１，以电穿孔法共转染大肠杆菌ＢＪ５１８３，获得重组腺病毒质粒。最后，将线性化的重组腺病毒质粒转染２９３细胞包装获得重组腺病毒，用荧光显微镜、ＰＣＲ及Ｗｅｓｔｅｒｎ ｂｌｏｔ分析ＬＩＧＨＴ基因的表达。 结果 用ＲＴ－ＰＣＲ法从人的ＰＢＭＣ中，扩增出７２３ ｂｐ的ｃＤＮＡ，测序证实为人ＬＩＧＨＴ基因。荧光显微镜、ＰＣＲ及Ｗｅｓｔｅｒｎ ｂｌｏｔ证实，ＬＩＧＨＴ重组腺病毒可感染２９３细胞，并在２９３细胞内进行有效的复制。结论成功地克隆了人ＬＩＧＨＴ基因，并构建了其重组腺病毒载体，为进一步研究ＬＩＧＨＴ基因的功能提供了条件。

人LIGHT胞外段基因的克隆及在大肠杆菌中的表达

目的:构建人LIGHT胞外段基因的原核表达载体,并在大肠杆菌中诱导表达。方法:从人外周血单核细胞来源的未成熟树突状细胞中提取总RNA,通过RT-PCR得到LIGHT胞外段基因,并将其克隆至pET32a(+)原核表达载体中,经双酶切鉴定及序列测定后的重组质粒转化入E.coli BL21,IPTG诱导表达目的蛋白,并用SDS-PAGE和Western blot进行检测。结果:RT-PCR扩增出了大小为543bp的LIGHT胞外段基因,经测序证明序列正确。SDS-PAGE和Western blot分析证实重组质粒可表达出Mr约为41000的蛋白。结论:成功地克隆了人LIGHT胞外段基因并在大肠杆菌中进行了表达为进一步研究LIGHT的功能打下了基础。

刺葡萄STS基因克隆及其在不同光质下的表达水平

【目的】研究刺葡萄芪合酶(STS)基因的序列特征及其在不同光质处理下的表达水平,旨在为进一步解析STS基因通过响应光信号调控刺葡萄愈伤组织中白藜芦醇合成的分子机制提供依据。【方法】以刺葡萄愈伤组织为材料,利用逆转录PCR(RT-PCR)技术成功克隆获得了4个STS基因,对这些基因及其编码蛋白进行了生物信息学分析,并利用实时荧光定量PCR(RT-qPCR)检测其在不同光质下和不同培养阶段的表达水平。【结果】成功地从刺葡萄愈伤组织中克隆获得了VdSTS5、VdSTS6、VdSTS20、VdSTS21共4个基因,均含1179 bp完整开放阅读框(ORF),编码392个氨基酸残基,预测其编码的是亲水性、无信号肽的细胞质定位蛋白,磷酸化修饰主要发生在苏氨酸和丝氨酸位点上,蛋白质的二级结构主要由α-螺旋、无规则卷曲和延伸链组成,STS与查尔酮合成酶(CHS)的蛋白质序列具有高度同源性。4个VdSTS的蛋白质序列在系统进化树上处于3个不同的大分支中,其中,VdSTS5与VdSTS21处在不同的分支中,而VdSTS6与VdSTS20聚在同一分支中,葡萄属不同种之间的STS蛋白质序列相似度高,推测其可能具有相同的功能。启动子顺式作用元件预测发现,4个VdSTS启动子含有多个光响应元件,还含有转录因子识别和作用元件、激素作用元件等。光质显著影响4个VdSTS的表达,在白光、红光、黄光和黑暗处理下,VdSTS的总体表达水平较高,而在绿光和紫光处理下,VdSTS的总体表达水平较低;同时,VdSTS对不同光质的响应不仅存在着基因间的差异,还存在不同培养阶段的差异,VdSTS5在培养后期强烈响应黄光的调控,VdSTS6、VdSTS20在培养中期对黑暗和红光的响应最强,VdSTS21则在培养中期强烈响应红光的调控,这些基因表达水平的变化影响着刺葡萄愈伤组织中白藜芦醇的合成。【结论】4个VdSTS对光质的响应存在差异,可能在响应不同光质调控中对刺葡萄愈伤组织白藜芦醇的合成起到重要作用。

火龙果HubHLH基因家族的全基因组分析及其对冬季补光诱导开花的表达响应

为了获得较完整的候选基因,探讨HubHLH基因在火龙果(Hylocereus undatus)冬季补光诱导开花过程的表达响应,对火龙果HubHLH基因家族进行全基因组分析。鉴定出153个HubHLH基因;这些基因的编码蛋白含有176~687个氨基酸,分子量大小为19.28~74.44 kDa,等电点(pI)为4.81~9.88,均为亲水蛋白,亚细胞定位预测大多定位到细胞核。为鉴定HubHLH基因家族的同源性,本研究将153个火龙果HubHLH和120个拟南芥AtbHLH蛋白进行系统发育比较分析。系统发育比较分析结果:火龙果HubHLH基因家族成员被分为12个组,25个亚族;对HubHLH基因家族的保守motif、基因结构及在染色体的位置分布的分析结果表明,同一亚族的基因具有相似的基序组成和基因结构。对HubHLH基因家族的内部复制事件的分析结果表明,有78条片段复制被鉴定为片段重复基因,说明片段复制是HubHLH基因家族的主要扩张力量。此外,基于已测定的关于火龙果冬季补光诱导开花的4个时期转录组数据,筛选到59个HubHLH基因在冬季补光诱导成花过程中有差异表达,随后对这59个HubHLH基因进行GO功能富集,发现它们在红光或远红光的反应、对光刺激的反应、有性生殖功能、对辐射的反应等功能上均有富集,说明HubHLH基因家族可能在冬季补光诱导火龙果成花过程中起到了调控作用。

MeLAZY1c基因调控木薯株型的初步研究

IGT基因家族参与作物株型的调控,LAZY属于IGT的亚家族。以拟南芥6个LAZY成员氨基酸序列为“种子”在木薯基因组中进行比对,在木薯中共鉴定到8个LAZY基因,其中MeLAZY1c与调控分枝角度的AtLAZY1高度同源。基于此,本研究以MeLAZY1c为研究对象,利用qRT-PCR分析发现MeLAZY1c在茎中转录水平最高,GUS染色显示pMeLAZY1c在维管束中染色较深。在MeLAZY1c启动子中发现8个光响应/调节元件,随后发现黑暗能显著抑制其表达水平。同时对MeLAZY1c进行基因编辑,获得纯合编辑株系19个,炼苗移栽后观测表型,发现melazy1c突变体植株与SC8野生型相比,其主茎呈现匍匐生长,并且弯曲部位茎外皮细胞形态扭曲变形且大小不一致,近地侧1 mm处细胞数目约是远地侧细胞数量的1.5倍,表明MeLAZY1c在木薯直立/匍匐生长建成方面发挥着重要作用。

LIGHT-Fc基因转染对食管癌细胞株Eca109抑制作用的初步研究

目的 探讨LIGHT对人食管癌细胞的体外抑制效应。方法 以DOTAP脂质体介导LIGHT Fc基因转染人食管癌细胞株Eca10 9,通过绘制细胞生长曲线及MTT比色法观察LIGHT Fc转染对Eca10 9细胞生长的影响 ,用RT PCR法检测LIGHT受体在Eca10 9细胞上的表达。结果 LIGHT Fc基因的表达可以抑制Eca10 9细胞的生长。在低血清培养时 ,Eca10 9 LIGHT细胞的生长曲线较对照组明显降低 ;MTT比色显示Eca10 9 LIGHT的细胞活力与对照组相比有显著性差异 (P<0 .0 5 )。结论 LIGHT Fc基因转染对人食管癌Eca10 9细胞具有体外抑制作用 。

人LIGHT胞外区基因的克隆及融合蛋白的表达

目的:克隆人LIGHT胞外区基因(hsLIGHT),构建其原核表达质粒,并诱导其在大肠杆菌中表达融合蛋白。方法:采用RT-PCR方法从HL60细胞中扩增hsLIGHT,将其克隆入原核表达质粒pGEX-4T-2,构建其重组表达质粒pGEX-4T- 2/hsLIGHT,以不同浓度IPTG诱导表达,于不同时间经SDS-PAGE和Western blot分析、鉴定。结果:经RT-PCR扩增获得的 hsLIGHT序列与Genebank报道的LIGHT基因胞外区序列完全一致;SDS-PAGE和Western blot分析证实重组质粒可表达出相对分子量为47 000的蛋白,与GST-hsLIGHT融合蛋白分子量一致。结论:成功完成了人LIGHT胞外区基因的克隆及其原核表达质粒的构建,在大肠杆菌E．coli BL21中经IPTG诱导表达了融合蛋白GST-hsLIGHT,为进一步探讨LIGHT的抗肿瘤生物学活性、探索肿瘤免疫治疗新方法奠定了基础。

不同波长LEDs光源对苦荞芽菜生长、生物活性物积累及抗氧化活性的影响

为探究不同波长LEDs光源对苦荞芽菜生长的影响,本研究以黑暗(D)作对照,采用单色红(R)、蓝(B)、绿(G)光源培养苦荞芽菜,收获后分别测定其形态指标、生物活性物积累量、类黄酮合成关键基因表达量及抗氧化活性指标,结果表明:B处理的苦荞芽菜下胚轴呈深红色,伸长生长受到强烈抑制,花青素、总黄酮及总酚积累量均最高,分别是D的7.18、2.96和2.49倍;R处理的下胚轴呈浅粉色,上述3种生物活性物积累量分别是对照的2.56、1.68和1.40倍;G处理的下胚轴伸长与R相似,外观呈透明白色,生物活性物积累量与对照无显著差异。B和R均强烈诱导类黄酮生物合成通路上关键结构基因表达,显著增加苦荞芽菜下胚轴抗氧化活性。此外,各单色光处理均未显著影响苦荞芽菜可食用部位的鲜重。由此可见,红、蓝LEDs光源可显著影响苦荞芽菜形态生长,并增加其生物活性物积累及抗氧化能力,以蓝光最佳。

不同光质组合的LED灯对小白菜生长及氮代谢生理机制的研究

以‘早熟五号’小白菜为试材,采用5种光质处理,研究不同光处理下小白菜生长、品质以及氮代谢主要相关酶活性及基因相对表达量的影响机理。与CK相比,其他4个处理均显著提高了小白菜的株高、茎粗、地上部分鲜质量、地上部分干质量、地下部分鲜质量和地下部分干质量。所有处理中,RBP处理下可溶性糖、可溶性蛋白、维生素C、铵态氮(NH4+-N)和硝态氮(NO3--N)含量达到最高,亚硝酸盐含量降低。RBP处理下硝酸还原酶(NR)活性和亚硝酸还原酶(NiR)活性最高,RB和RBP处理明显增加了谷氨酸合成酶(GOGAT)活性。RB、RBG和RBP处理下谷氨酰胺合成酶(Gs)活性显著高于CK,谷氨酸脱氢酶(GDH)活性则在RBP处理下最低。基因表达方面,RBP处理下NR、NiR、Gs和GDH的mRNA表达水平最高,GOGAT的mRNA表达量以RB处理最高,RBP处理其次。结合生长量、氮代谢和相关基因表达的综合表现,在红蓝光基础上增加紫光对小白菜的生长最为有利。

人LIGHT基因的克隆及其在293T细胞中的表达

目的 克隆人LIGHT分子的全长cDNA ,构建重组真核表达质粒pCI neo LIGHT并在 2 93T细胞上获得稳定表达。方法 从人T细胞cDNA文库中用PCR技术克隆人LIGHT全长cDNA ,装入T Easy载体 ,测序证实后 ,将LIGHTcDNA装入质粒pCI neo中构建真核表达载体。用电穿孔法转染 2 93T细胞 ,经G418筛选后 ,用流式细胞仪检测LIGHT分子的表达。结果 测序证实克隆的LIGHT全长cDNA阅读框正确完整 ,酶切证实LIGHT pCI neo中LIGHT插入方向正确。转染的2 93T细胞经G418筛选 3个月后 ,流式细胞仪检测有 78 69%的细胞表达人LIGHT分子。结论 成功克隆LIGHT基因并获得稳定表达膜型LIGHT分子的 2 93T细胞系。

人LIGHT基因重组慢病毒的构建以及在脐血间质干细胞中的表达

目的构建带有人LIGHT基因的慢病毒载体,观察其在人脐血间质干细胞中的表达。方法通过逆转录聚合酶链反应从PCD DNA-LIGHI、质粒中获得人LIGHT基因,利用infusion技术重组构建慢病毒载体质粒pGC-FU-LIGHT,在脂质体lipofectamine 2000介导下与结构质粒pHelper1.0及包膜蛋白质粒pHelper2.0共转染293T细胞包装生产慢病毒。将人脐血间质干细胞分为实验组(pGC-FU-LIGHT)、空载体对照组(pGC-FU-EGFP)及空白组(脐血间质干细胞),分别用重组慢病毒、空载慢病毒、PBS感染后,采用RT-PCR以及Elisa检测IIGHT表达情况。结果所获LIGHT基因经测序后与Gene Bank报道序列完全一致;重组慢病毒载体质粒pGC-FU-LIGHT经鉴定正确;三质粒共转染293T细胞成功,收集、浓缩病毒后测定其滴度为2×107TUJ/L,感染UJCBMSCs后RT-PCR、Elisa检测各组细胞均有LIGHT的表达,其中试验组pGC-FU-LIGHT组更大最表达LIGHT,与其余2组(pGC-FU-EGFP、UJCBMSCs)比较差异具有统计学意义。结论成功构建带有LIGHT基因的慢病毒载体并实现在脐血间质干细胞中的表达,为间充质干细胞移植治疗胃癌的应用奠定了基础。

强光胁迫对转玉米C_(4)型ZmPEPC+ZmPPDK基因小麦光合和生理特性的影响

为研究转玉米C_(4)型PEPC(磷酸烯醇式丙酮酸羧化酶基因)和PPDK(丙酮酸磷酸双激酶基因)双基因小麦对强光胁迫的光合和生理响应,以转ZmPEPC+ZmPPDK基因小麦株系PCK30和PCK60及其野生型对照材料(WT)为试验材料,鉴定了外源基因在转基因小麦中的表达量,在抽穗期和灌浆期测定正常光强(NL)和强光胁迫(HL)处理下转基因小麦的光合酶活性、叶绿素含量、气体交换参数、叶绿素荧光参数、活性氧物质含量和抗氧化酶活性。结果表明,2个转基因株系在转录水平上高效表达了PEPC和PPDK基因。在不同时期NL和HL处理下,转基因小麦的PEPC、PPDK、NADP-ME和Rubisco的酶活均显著高于WT,且HL处理下高出WT的幅度更明显。与NL处理相比,转基因小麦和WT的叶绿素含量在HL处理下显著降低,但转基因株系的下降幅度更小,且HL处理下转基因株系的叶绿素含量显著高于WT。两种水平处理下,转基因小麦PCK30、PCK60的净光合速率(P_(n))均显著高于WT,且HL处理下高出幅度更明显,抽穗期增幅分别为15.26%和17.57%,灌浆期为13.41%和15.82%。气孔导度、F_(v)/F_(m)、q_(p)的变化趋势与P_(n)一致,胞间二氧化碳浓度的变化趋势与P_(n)相反。转基因株系在HL处理下产生的活性氧物质和丙二醛含量显著低于WT,而抗氧化酶变化趋势与之相反。连续2年田间小区产量试验,转基因小麦PCK30和PCK60平均比WT高8.37%和10.16%。PEPC和PPDK在小麦中的过表达增强了小麦内源的光合酶、光化学效率和抗氧化酶活性,增强了强光下的叶片细胞膜的稳定性,保护了光合机构,维持了较高的光合效率,从而提高了转基因小麦的耐强光胁迫能力。

小桐子早期光诱导蛋白基因ELIP克隆及其与靶向miRNA的互作与低温下共表达分析

【目的】作为一种喜温冷敏植物,低温严重影响小桐子的生长发育、地域分布与产量。前期工作发现12℃低温锻炼可显著提高小桐子的抗冷性,小桐子早期光诱导蛋白(ELIP)基因是低温高响应基因。为探究JcELIP在小桐子低温响应中的作用,全面了解JcELIP的结构、调控机制、进化关系及JcELIP与miRNAs的互作关系,也为后续小桐子抗冷分子育种提供一个重要的候选基因资源。【方法】通过RT-PCR克隆了小桐子JcELIP基因并对其进行系统的生物信息学分析,采用RT-qPCR分析JcELIP基因在根、茎、叶中及12℃低温锻炼过程中的表达变化,鉴定与JcELIP互作的miRNAs,进行了12℃低温锻炼过程中的共表达分析。【结果】该基因完整的开放阅读框(ORF)为585 bp,编码194个氨基酸,蛋白的分子质量为2.04 kD,理论等电点为9.59,属于稳定的疏水碱性蛋白,具有3个疏水跨膜螺旋;三级结构主要由ɑ-螺旋和无规则卷曲组成,具有叶绿素a/b结合位点。顺式作用元件预测显示JcELIP具有脱落酸等激素响应元件。进化分析显示:小桐子JcELIP与木薯MeELIP同源性最高。RTqPCR分析显示:正常生长条件下小桐子JcELIP在根、茎、叶中的表达量无显著差异;12℃冷锻炼条件下JcELIP在叶中表达快速上调,48 h时其表达量达到对照组的64.8倍,表明JcELIP参与了小桐子对冷胁迫的响应与适应。基于小桐子降解组数据,鉴定到miR390-x、miR6476-x和novel-m0090-3p等8个miRNAs对JcELIP的表达具有调控作用;共表达分析结果表明在12℃低温锻炼过程中JcELIP的表达受miR390-x和novel-m0090-3p显著负调控。【结论】JcELIP高响应低温胁迫并与miRNAs互作,表明其在小桐子响应低温胁迫过程中发挥重要作用。