人工改造野生大豆GsDREB2基因对植物耐盐和耐渗透胁迫能力的影响

DREB (dehydration responsive element binding protein)转录因子是一个干旱应答元件的结合蛋白,它能特异结合启动子中含有DRE/CRT顺式元件,激活逆境诱导基因的表达,调控植物对干旱、低温、高盐、高温等胁迫的耐逆性。大量研究表明DREB转录因子在信号传导、作用机理及基因表达方面存在复杂性。为了野生大豆来源GsDREB2基因能更有效地发挥功能,人工突变该基因的负向调节结构域(negative regulatory domain, NRD,140~204),经改造命名为GsDREB2-mNRD。在酵母中比较全长基因(FLDREB2)和GsDREB2-mNRD转录激活和与DRE元件结合的能力,并验证GsDREB2-mNRD核定位情况。分别将FLDREB2和GsDREB2-mNRD转化拟南芥,通过拟南芥幼苗期盐胁迫和渗透胁迫试验,比较GsDREB2-mNRD和FLDREB2在提高植物耐盐和渗透胁迫方面的差异。结果表明,GsDREB2基因内部存在着负向调节结构域(NRD),抑制了GsDREB2转录激活功能和DRE元件结合的特性;经改造的GsDREB2基因依然能定位在细胞核;超量表达GsDREB2-mNRD基因的拟南芥耐盐和渗透胁迫能力明显强于非转基因对照,也高于FLDREB2基因超表达的拟南芥;野生大豆来源的GsDREB2基因NRD结构域的缺失可增强该基因在植物耐盐、渗透胁迫等逆境胁迫下的功能。     

低温胁迫对狗牙根生理及基因表达的影响

以耐寒性差异较大的杂交狗牙根品种运动百慕大、天堂419和普通狗牙根品种保定狗牙根为试验材料,分析了昼夜温度为适温(30 ℃/25 ℃)、亚适温(18 ℃/12 ℃)、冷害(8 ℃/4 ℃)和冻害(4 ℃/-4 ℃)4种温度处理下,低温对狗牙根生长速率和草坪质量、MDA含量、叶绿素荧光(F_v/F_m)、光合作用(P_n、G_s、C_i、T_r)、H₂O₂和O2·-含量、抗氧化酶活性(SOD、POD、CAT、APX)、抗氧化酶及耐寒相关基因(CBF1、COR、LEA)表达的影响。结果表明,随着温度的降低,狗牙根草坪质量、生长速率、F_v/F_m和光合作用显著下降,但在耐寒性强的运动百慕大中下降幅度较小;低温下叶片MDA和H₂O₂含量及O2·-产生速率显著升高,其中在耐寒性弱的保定狗牙根中升高程度更大;狗牙根叶片抗氧化酶活性及抗氧化酶基因的表达水平随着处理温度的下降而显著升高,其中在运动百慕大中更为明显;狗牙根CBF1、COR和LEA基因表达量随着温度的下降而显著升高,尤其是在耐寒狗牙根运动百慕大中更为显著。低温诱导的抗氧化酶和耐寒相关基因上调表达,增强了细胞内的抗氧化防御,有助于狗牙根植株维持较高的光合作用和细胞膜稳定性,延缓叶片的衰老,从而提高了狗牙根的抗寒能力。     

Differential gene expression of Eph‐ephrin A1 and LEPR‐LEP with high or low number of embryos in pigs during implantation

The objective of this study was to ascertain whether mRNA and protein expressions of implantation‐related genes (erythropoietin‐producing hepatocellular receptor–ligand A1, Eph‐ephrin A1 and leptin receptor–leptin, LEPR‐LEP) differed between pigs with high and low number of embryos, and whether these differences in gene expression might affect embryo implantation. Experimental pig groups (n = 24) for high and low number of embryos were prepared by altering the number of eggs ovulated in pre‐pubertal gilts treated with 1.5 × (High) or 1.0 × (Low) PG600 ([400 IU PMSG + 200 IU hCG]/dose, AKZO‐NOBEL). Gilts expressing oestrus were artificially inseminated twice and maintained in breeding and gestation until the reproductive tract was collected on day 22 of pregnancy. At slaughter, the reproductive tracts from each pregnant gilt from each treatment were immediately processed to collect samples for RNA and protein analysis. Within each gilt, three conceptus points were sampled, one from each horn and then a random conceptus within the tract. At each conceptus point, endometrial attachment site, chorion–allantois and embryo were collected and immediately frozen in liquid nitrogen. Number of corpus luteum (CL) (35.4 vs. 12.6) and total embryo number (18.8 vs. 10.2) were greater in the high‐embryo compared to the low‐embryo group, respectively (p < .05). Real‐time qPCR results showed that Eph‐ephrin A1 mRNA expression was less in the high‐embryo (p < .05) compared to the low‐embryo group. In addition, Western blotting analysis indicated that Eph‐ephrin A1 and LEP protein expression at endometrial attachment site in high‐embryo was less (p < .05) compared to low‐embryo group. It was also noted that mRNA expression of Eph‐ephrin A1 and LEPR‐LEP was greater in pregnant than non‐pregnant gilts (p < .05). Moreover, mRNA expression of Eph‐ephrin A1 (p < .05) and LEPR‐LEP was greatest at endometrial attachment site among all three tissues. There was a positive correlation between expressions of Eph‐ephrin A1, LEPR‐LEP and embryo length with the correlation coefficient 0.31–0.59. For Eph‐ephrin A1, the highest correlation coefficient appeared between Eph A1 expression and normal embryo number, between ephrin A1 expression and embryo length. For LEPR‐LEP, the highest correlation coefficient appeared between LEPR‐LEP expression and ovary weight (0.79 for both, p < .05), followed by embryo length and weight. The results of this study suggest that low expression of Eph‐ephrin A1 and LEPR‐LEP is somehow related to increased embryo number during implantation and that endometrial attachment site might be the main target tissue of these gene products. Yet, the increased expression of Eph‐ephrin A1 and LEPR‐LEP appeared associated with increased embryo growth (length and weight) and ovary weight, Eph‐ephrin A1 and LEPR‐LEP might play roles in the regulation of embryo implantation in pigs.     

Effects of five cryoprotectants on proliferation and differentiation‐related gene expression of frozen‐thawed bovine calf testicular tissue

The cryopreservation of testicular tissue is a potential method for preserving male fertility. However, the effect of cryopreservation on bovine calf testicular tissue is scarce. This study investigated the effect of different cryoprotectants on bovine calf testicular tissue at the molecular level. Testicular tissue from ten immature bovine calves (6 months) was collected after slaughter and cryopreserved in an extender containing different concentrations of the following five cryopreservation solutions (CP): bovine serum albumin (BSA) with 5% dimethyl sulfoxide (DMSO), trehalose with 5% DMSO, DMSO and glycerol and ethylene glycol (EG). After 7‐day cryopreservation, the expression levels of three spermatogonial stem cell (SSC)‐related genes, octamer‐4 (OCT4), KIT ligand (MGF/SCF) and kit oncogene (C‐KIT), were investigated by quantitative PCR (qPCR). The cell viability was highest for the tissues preserved with 30 mg/ml BSA (77.82% ± 1.22) and 40 mg/ml trehalose (74.23% ± 1.16) compared with other groups (p < 0.05), and the level of expression of the three genes was highest with 30 mg/ml BSA (p < 0.05). Compared with other CPs, the 30 mg/ml BSA and 40 mg/ml trehalose have the better cryopreserve protection. The 30 mg/ml BSA is the most viable media for the cryopreservation of testicular tissue from cattle.     

Identification of organic acid-related genes and their expression profiles in two pear (Pyrus pyrifolia) cultivars with difference in predominant acid type at fruit ripening stage

‘Yandangxueli’ is a pear cultivar with predominant citric acid in the ripe fruit, different from most of pear cultivars such as ‘Gengtouqing’ in which malic acid is the predominant acid type. It was found that ‘Yandangxueli’ accumulated citric acid for three times against that in ‘Gengtouqing’ at fruit ripening stage. To investigate the mechanism of citric acid accumulation in ‘Yandangxueli’, organic acids content, gene expression and enzyme activity were studied in both cultivars. Five genes, Pp:mtCs, Pp:cyAco, Pp:cyIdh, Pp:mtMdh and Pp:cyMe which encoded citric synthase (CS), cytosolic aconitase (cyACO), NADP-dependent isocitrate dehydrogenase (NADP-IDH), NAD-dependent malate dehydrogenase (NAD-MDH) and NADP-dependent malic enzyme (NADP-ME) respectively, were identified from pear fruit. Their expression profiles and the corresponding enzyme activities were determined throughout fruit development in both cultivars. Results from these enzymes indicated that there were no strict relationship between gene expression, enzyme activity and citric acid accumulation. Expression analysis for two Py:vVAtp genes encoding vacuolar H⁺-ATPase A subunit and one Py:vVpp gene encoding Vacuolar H⁺-pyrophosphatase showed that they were all with up-regulated expression at the later development stage of ‘Yandangxueli’ but with down-regulated expression in ‘Gengtouqing’. Therefore, it is concluded that the different ability in citric acid transportation and storage might be involved in the high citric acid content in ‘Yandangxueli’.     

花生抗黄曲霉相关基因PnLOX2的原核表达及RNAi载体的构建与转化

本研究对利用基因芯片技术从抗、感黄曲霉花生品种中分离出的差异表达基因PnLOX2进行原核表达,得到分子量为121.5 kD的融合表达产物;构建了PnLOX2基因的3'端反向重复结构并转化花生,得到了转基因花生苗,为反向鉴定PnLOX2基因在花生抗黄曲霉侵染过程中的功能奠定基础。     

青花菜锌指蛋白基因BoCCCH1的克隆和表达分析

CCCH锌指蛋白广泛存在于真核生物中,在植物的生长、发育和逆境胁迫响应中起着重要作用。为获知青花菜CCCH转录因子基因的序列特征和表达特性,从青花菜叶片中克隆到1个CCCH基因,命名为Bo CCCH1,同时,利用RT-PCR方法研究了其在不同器官及霜霉菌侵染叶片中的表达模式。序列分析结果表明,Bo CCCH1的基因组全长为1 568 bp,包含1个长度为599 bp的内含子,2个长度分别为627 bp、342 bp的外显子,编码322个氨基酸,蛋白质的分子量与等电点分别为35 296.42 Da和8.47;Bo CCCH1有2个锌指结构,类型均为C-X8-C-X5-C-X3-H。系统发育分析结果表明,Bo CCCH1与其它植物的21条同源序列在进化树上分为6组,来自十字花科的CCCH与Bo CCCH1处于同一分支。基因表达结果表明,Bo CCCH1在叶、花茎、嫩角果、花蕾和花中表达,其中花茎和嫩角果的表达量最高,但在根中未检测到表达;在霜霉菌侵染下,叶片Bo CCCH1的表达量升高,到24 h和36 h时达到最大,推测Bo CCCH1与霜霉病抗性相关。本研究为进一步探明Bo CCCH1在霜霉病抗性反应中的功能奠定了基础。