Genome-Wide Identification of Aquaporin Gene Family in Pitaya Reveals an HuNIP6;1 Involved in Flowering Process

Aquaporins (AQPs) are essential membrane proteins involved in seed maturation and germination, stomata movement, photosynthesis, and regulation of plant flowering processes. Pitaya flowers are open at night and wither at daybreak, which shows an obvious circadian rhythm. In this study, a comprehensive genome-wide analysis of AQPs in Hylocereus undantus was conducted to screen key genes associated with flowering processes. A total of 33 HuAQP genes were identified from the H. undantus genome. The 33 HuAQPs were grouped into four subfamilies: 10 PIPs, 13 TIPs, 8 NIPs, and 2 SIPs, which were distributed on 9 out of 11 pitaya chromosomes (Chr) (except for Chr7 and Chr10). Results from expression profiles showed that HuNIP6;1 may be involved in pitaya’s floral opening. HuNIP6;1 was localized exclusively in the cell membrane. Overexpression of HuNIP6;1 in Arabidopsis thaliana significantly promoted early flowering through regulating negative flowering regulators of MJM30, COL9, and PRR5, suggesting that HuNIP6;1 plays key roles in regulating flowering time. The present study provides the first genome-wide analysis of the AQP gene family in pitaya and valuable information for utilization of HuAQPs.     

Differences in Leaf Morphology and Related Gene Expression between Diploid and Tetraploid Birch (Betula pendula)

Plant polyploidization changes its leaf morphology and leaf development patterns. Understanding changes in leaf morphology and development patterns is a prerequisite and key to studying leaf development in polyploid plants. In this study, we quantified and analyzed the differences in leaf morphology, leaf growth polarity, and leaf size between diploid and tetraploid birches (Betula pendula subsp. pendula), and preliminarily investigated genes involved in leaf growth and development in birch. The results showed significant changes in leaf morphology in tetraploid birches, especially the basal part of the leaf. In addition, the proximal growth rate of tetraploid leaves was altered. The changed proximal growth rate did not affect the growth polarity pattern of tetraploid leaves. The leaf area of tetraploid was significantly larger than that of diploid birch. The difference in leaf size was mainly due to differences in their growth rates in the middle and late stages of leaf development. Increased cell expansion capacity was the major reason for the enormous leaves of tetraploid birch; however, cell proliferation did not contribute to the larger tetraploid leaf. The gene expression of ATHB12 was associated with cell size and leaf area, and may be a critical gene affecting the leaf size in diploid and tetraploid birches. The results will provide valuable insights into plant polyploid leaf development and a theoretical basis for later investigations into the molecular mechanisms underlying the gigantism of tetraploid birch leaves.     

小鼠鸟氨酸脱羧酶抗酶1功能基因的克隆及原核表达

目的克隆小鼠鸟氨酸脱羧酶抗酶1(OAZ1)功能基因,原核表达并纯化OAZ1重组蛋白。方法采用RT-PCR法从小鼠黑色素瘤细胞总RNA中扩增OAZ1基因,通过重叠延伸PCR技术构建无需移码即可全长翻译的功能基因,并构建重组表达质粒pET15b/OAZ1-T,转化大肠杆菌BL21(DE3),经IPTG诱导表达。表达的重组蛋白经Ni2+-NTA亲和层析纯化后,进行SDS-PAGE和Western blot分析。结果重叠PCR法扩增出692bp的OAZ1功能基因,重组表达质粒经酶切及测序鉴定正确,重组蛋白可在大肠杆菌中以包涵体形式高效表达。纯化的重组蛋白纯度可达79.96%,且可与抗His标签抗体特异性结合。结论已成功克隆了小鼠OAZ1功能基因,原核表达并纯化了重组OAZ1蛋白。     

JTV1基因真核表达质粒的构建及其在K562细胞中的表达

目的 构建JTV1基因真核表达质粒并稳定转染人白血病细胞系K562,检测转染细胞中JTV1基因mRNA和蛋白的表达水平及其对K562细胞增殖的影响。方法从人外周血单个核细胞中克隆JTV1基因,并将其插入pcDNA3.1表达载体中,构建真核重组表达质粒pcDNA3.1-JTV1,经脂质体介导转染K562细胞,采用RT-PCR和Western blot法鉴定转染细胞中JTV1基因mRNA和蛋白的表达水平;MTT法检测JTV1稳定表达对K562细胞增殖的影响。结果重组表达质粒pcDNA3.1-JTV1经双酶切及测序证实,目的基因已插入质粒中;人JTV1基因能在K562细胞中稳定表达;JTV1具有抑制K562细胞增殖的作用。结论已成功构建了JTV1基因真核表达质粒,并获得了稳定表达人JTV1基因的K562细胞克隆,为进一步研究人JTV1基因的功能及其与白血病细胞增殖及凋亡的相关性提供了细胞模型。     

Inhibition of Mammalian Target of Rapamycin Complex 1 (mTORC1) Downregulates ELOVL1 Gene Expression and Fatty Acid Synthesis in Goat Fetal Fibroblasts

Elongation of very-long-chain fatty acids 1 (ELOVL1) is a ubiquitously expressed gene that belongs to the ELOVL family and regulates the synthesis of very-long-chain fatty acids (VLCFAs) and sphingolipids, from yeast to mammals. Mammalian target of rapamycin complex 1 (mTORC1) is a central regulator of cell metabolism and is associated with fatty acids synthesis. In this study, we cloned the cDNA that encodes Cashmere goat (Capra hircus) ELOVL1 (GenBank Accession number {"type":"entrez-nucleotide","attrs":{"text":"KF549985","term_id":"558615228"}}KF549985) and investigated its expression in 10 tissues. ELOVL1 cDNA was 840 bp, encoding a deduced protein of 279 amino acids, and ELOVL1 mRNA was expressed in a wide range of tissues. Inhibition of mTORC1 by rapamycin decreased ELOVL1 expression and fatty acids synthesis in Cashmere goat fetal fibroblasts. These data show that ELOVL1 expression is regulated by mTORC1 and that mTORC1 has significant function in fatty acids synthesis in Cashmere goat.     

Changes of Sensory Quality,Flavor-Related Metabolites and Gene Expression in Peach Fruit Treated by Controlled Atmosphere (CA) under Cold Storage

Controlled atmosphere (CA) has been used to alleviate chilling injury (CI) of horticultural crops caused by cold storage. However, the effects of CA treatment on peach fruit sensory quality and flavor-related chemicals suffering from CI remain largely unknown. Here, we stored peach fruit under CA with 5% O₂ and 10% CO₂ at 0 °C up to 28 d followed by a subsequent 3 d shelf-life at 20 °C (28S3). CA significantly reduced flesh browning and improved sensory quality at 28S3. Though total volatiles declined during extended cold storage, CA accumulated higher content of volatile esters and lactones than control at 28S3. A total of 14 volatiles were positively correlated with consumer acceptability, mainly including three C6 compounds, three esters and four lactones derived from the fatty acid lipoxygenase (LOX) pathway. Correspondingly, the expression levels of genes including PpLOX1, hyperoxide lyase PpHPL1 and alcohol acyltransferase PpAAT1 were positively correlated with the change of esters and lactones. CA elevated the sucrose content and the degree of fatty acids unsaturation under cold storage, which gave us clues to clarify the mechanism of resistance to cold stress. The results suggested that CA treatment improved sensory quality by alleviating CI of peach fruits under cold storage.     

HIV-1 HXB2株ta(tB41-101N)融合基因原核表达质粒的构建及表达

目的构建HIV-1HXB2株ta(tB41-101N)融合基因原核表达质粒,表达并纯化Ta(tB41-101N)融合蛋白,为进一步研究其免疫原性奠定基础。方法在HIV-1HXB2株天然Tat蛋白N-端添加Tat41-101位氨基酸(aa),采用PCR法从HIV-1HXB2株tat基因中扩增分别编码Tat41-101aa和Tat1-101aa的tat41-101和tat1-101两个基因片段,重叠延伸PCR法扩增其融合基因ta(tB41-101N),并构建其原核表达质粒pET32a-ta(tB41-101N),经双酶切及测序验证后,转化大肠杆菌BL21(DE3),IPTG诱导表达。融合蛋白经Ni2+-NTA柱亲和层析纯化后,进行SDS-PAGE、Westernblot及ELISA鉴定。结果重叠延伸PCR扩增出约500bp的ta(tB41-101N)融合基因,酶切及测序结果表明重组表达质粒pET32a-ta(tB41-101N)构建正确。SDS-PAGE分析显示,表达的Ta(tB41-101N)融合蛋白相对分子质量约为36000,约占菌体总蛋白的7%,纯化后纯度约为60%;Westernblot和ELISA分析显示,Ta(tB41-101N)融合蛋白与小鼠抗Tat单抗及抗-HIV阳性血清(抗Tat抗体阳性)均呈特异性阳性反应。结论已成功构建了HIV-1HXB2株ta(tB41-101N)融合基因原核表达质粒,并表达了其融合蛋白,该蛋白较好地保留了天然Tat蛋白的免疫反应性,为Tat新型疫苗的研究奠定了基础。     

HIV-1 gag和env基因片段的构建及表达

为研究人免疫缺陷病毒（HIV－1）的分子生物学，发展艾滋病的诊断试剂，克隆了含HIV－1env和gag基因cDNA的质粒pUCF12。用PvuⅡ和HincⅡ降解pUCF12质粒，得到含部分p17、全部p24和全部p15基因的cDNA片段，用BglⅡ降解pUCF12质粒得到含有部分gp120和全部gp41的cDNA片段。将该两个片段分别亚克隆到表达载体PGex上，在pTac启动子的调控下，部分gag基因和env基因在大肠杆菌中表达．在SDS－PAGE中，GST＋gag融合蛋白呈现一条约70kD的染色带，其表达水平约占菌体总蛋白的18％．而GST－env蛋白则呈现一条约65kD的蛋白带，该表达蛋白约占菌体总蛋白的15％。经免疫印迹分析，该两种表达产物可分别与抗-P24和抗-gP41的单克隆抗体反应，用Abbott试剂也证明为HIV－1抗原蛋白。     

鳗弧菌W-1金属蛋白酶基因真核表达载体的构建及其在动物细胞中的表达

采用基因克隆方法,以带有突变的鳗弧菌W-1金属蛋白酶基因的原核表达质粒pET24d(+)/m-empA7为模板,通过PCR扩增得到m-empA7基因,将其插入pCDNA3.1(+)构建重组真核表达质粒pCDNA3.1(+)/m-empA7;然后用磷酸钙法将其转染入动物细胞CHO和HEK293T中并进行瞬时表达,分别以RT-PCR和Western-blot检测重组质粒的基因转录和蛋白表达情况。结果表明,成功将突变的鳗弧菌W-1金属蛋白酶基因转染至真核细胞中,并能够在动物细胞中正确地转录和表达,为鳗弧菌基因工程疫苗的研制奠定了基础。     

幽门螺杆菌粘附素hpa A和霍乱毒素B亚单位ctx B融合基因的原核表达

目的　构建幽门螺杆菌粘附素 (hpaA)和霍乱毒素B亚单位 (ctxB)融合基因的原核表达载体 ,诱导表达并进行纯化。方法　用PCR扩增hpaA和ctxB两个目的基因片段 ,克隆至同一pQE 30表达载体中 ,构建含双基因的表达质粒pQE hct,转化E .coliDH5α ,经IPTG诱导表达融合蛋白HCT ;经Westernblot分析其免疫原性 ,采用镍离子柱进行纯化。结果　经测序HCT融合基因片段由 116 1bp组成 ,为编码 387个氨基酸残基的多肽。经SDS PAGE分析相对分子质量约为 4 0 0 0 0。可溶性蛋白占菌体总蛋白的 2 5 %以上 ,经亲和层析后可获得纯度为 92 %以上的重组融合蛋白。经Westernblot检测可被Hp全菌抗血清和CT抗血清识别。结论　已成功构建融合蛋白HCT的原核表达载体并能高效表达 ,为研制Hp口服疫苗提供依据