马铃薯AP2/ERF转录因子的克隆及植物表达载体构建

AP2/ERF基因家族转录因子普遍存在于植物中,参与植物体内的各种生物学过程,包括植物的生长发育、生物和非生物胁迫响应等。前期转录组测序的结果发现马铃薯‘加湘1号’一个AP2/ERF家族基因(PGSC0003DMG400012154)在接种晚疫病菌(Phytophthora infestans)24 h后被显著激活。从接种P.infestans 24 h的‘加湘1号’的总RNA中通过RT-PCR获得了该基因CDS序列为885 bp,BLAST分析显示该基因编码一个295个氨基酸残基的蛋白,并且含有1个AP2/ERF结构域,是AP2/ERF转录因子家族中的ERF亚家族的一员。本研究将该基因与XcmⅠ酶切的表达载体pCXSN连接,转化大肠杆菌,通过测序挑选插入正确克隆酶切验证,并成功转化农杆菌。本研究结果为进一步研究该基因的功能提供了帮助。     

白屈菜CmFAD2基因的克隆及植物转化载体构建

通过对白屈菜低温应答过程的转录组分析发现膜脂不饱和化相关基因的表达在一定过程中发生变化,脂肪酸去饱和酶基因FAD2在随温度的变化趋势为正"V"型,且表达量变化显著。利用NCBI等在线软件对序列进行相关生物学信息分析,并对白屈菜FAD家族成员FAD2基因的完整开放阅读框(ORF)进行克隆,并命名为CmFAD2。选用克隆载体pMD-19-T,转化大肠杆菌DH5α,测序验证序列正确性及完整性。将目的基因与植物表达载体pRI-201-AN连接构建重组DNA pRI-201-AN-Cm FAD2,电击法转化农杆菌LBA4404,利用菌液PCR法验证成功。该基因可作为药用植物抗寒品种创制的候选基因。     

寨卡病毒SYBR GreenⅠreal-time PCR方法的建立及应用

为建立一种针对寨卡病毒的快速诊断方法,本研究根据寨卡病毒的3’端保守基因序列,设计合成1对引物,建立了检测寨卡病毒的荧光定量PCR方法。结果显示:所建立的检测方法的Ct值与标准品在1.41×10^1~1.41×10^10^ copies/μL具有良好的线性关系,相关性为1,斜率为-3.502;灵敏性结果显示,该方法的检测限度为1.41×10^1 copies/μL,是普通PCR的10000倍;特异性结果显示,对CHIKV、DENV和JEV无特异性扩增,特异性强;重复性试验结果显示,组内和组间变异系数均小于1%,重复性好。本研究建立的SYBR Green I real-time PCR检测方法,可用于寨卡病毒感染的快速诊断。     

Milk vetch dwarf virus infection in the Solanaceae and Caricaceae families in Southeast Asia

Milk vetch dwarf virus (MDV) is an important member of the genus Nanovirus and is transmitted by the aphid Aphis craccivora. MDV has multiple single-stranded DNA genome components, each approximately 1 kb, and two or three alpha-satellite molecules. It mainly infects plants of the legume family Fabaceae. Recently, papaya (Carica papaya) collected in Yesan, South Korea, displaying symptoms of leaf yellowing and dwarfism, was identified as a new host for MDV. To examine the geographical distribution of MDV, papaya samples with symptoms were harvested in South Korea, Vietnam, and Taiwan in August 2018, along with tomato and pepper samples grown in adjacent fields in Vietnam. The results revealed the presence of MDV not only in papaya but also in pepper and tomato. This MDV infection in members of the Solanaceae family was confirmed by Southern blot hybridization performed using a PCR product of segment S as a probe. Based on sequence analysis of three MDV components (M, S, and C3), we verified the presence of three different isolates of MDV in these three countries and homology between sequences of isolates from papaya and from members of the Solanaceae from Vietnam. Taken together, our results clearly demonstrate MDV infection in Vietnam and Taiwan for the first time and confirm that MDV can infect economically important pepper and tomato.     

Leaf carbohydrates assimilation and metabolism affect seed yield of rapeseed with different waterlogging tolerance under the interactive effects of nitrogen and waterlogging

Waterlogging is a main factor causing rapeseed (Brassica napus L.) yield loss, and reasonable nitrogen (N) applications can compensate for this loss. To investigate the effects of N rates on seed yield of waterlogged rapeseed, the waterlogging-tolerant rapeseed variety ZS 9 and sensitive variety GH01 were waterlogged for 0 and 10 days with five leaves at the seedling stage under four N rates (0, 90, 180 and 270 kg/ha). Waterlogging significantly decreased seed yield, while N application can alleviate the yield loss. The yield decrease rate of waterlogged GH01 was greater than that of ZS 9 under the same N rate. During the seedling and bolting stage, the leaf photosynthetic rate (Pn) and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity increased, while activities of adenosine diphosphate glucose pyrophosphorylase (AGPase), sucrose synthase (SuSy) and sucrose phosphate synthase (SPS) decreased with more N under the same watering conditions. Compared to the plants without waterlogging, the leaf Pn and Rubisco activity, starch and sucrose contents of waterlogged rapeseed decreased at the two stages; activities of AGPase, SuSy and SPS of waterlogged rapeseed decreased at the seedling while increased at the bolting stage for both the two varieties. At the flowering stage, the Pn, the activities of Rubisco, AGPase, SuSy, SPS and contents of sucrose, starch increased with more N application for both ZS 9 and GH01. Compared to the plants without waterlogging, the Pn and Rubisco activity for waterlogged plants of the two varieties increased; the waterlogged plants of tolerant variety had higher activities of AGPase, SuSy and SPS, while those of sensitive variety was significantly lower. However, the decreased starch and sucrose content were found in both tolerant and sensitive varieties. The activities of AGPase, SuSy and SPS at flowering were highly positively correlated with yield under the interactive effects of N and waterlogging. These results suggested that the flowering stage is the most important stage that N had the positive regulation on waterlogged rapeseed growth. The carbohydrates translocation from leaves to seeds of the tolerant variety were enhanced after waterlogging, while that of the sensitive variety was still inhibited. This was the main reason for the difference in yield between the two waterlogged varieties.     

利用酵母双杂交系统筛选与草莓镶脉病毒移动蛋白P1互作的寄主因子

 构建感染草莓镶脉病毒(SVBV)森林草莓的酵母cDNA文库,利用酵母双杂交系统,筛选出与SVBV P1蛋白互作的15种寄主因子。生物信息学分析发现,这15种寄主因子参与茉莉酸途径、泛素化、光合作用、抗病抗逆、蛋白修饰、蛋白运输和氧化还原等多种生物过程。另外,这些寄主因子还具有其他分子功能,包括氧化还原酶活性、蛋白二硫化物异构酶活性和金属离子结合活性等。本研究初步探讨了P1与寄主因子的互作机理,为揭示SVBV侵染森林草莓以及SVBV在寄主中扩展的分子机制提供理论依据。     

Protective efficacy of an H5/H7 trivalent inactivated vaccine produced from Re-11, Re-12, and H7-Re2 strains against challenge with different H5 and H7 viruses in chickens

We developed an H5/H7 trivalent inactivated vaccine by using Re-11, Re-12, and H7-Re2 vaccine seed viruses, which were generated by reverse genetics and derived their HA genes from A/duck/Guizhou/S4184/2017(H5 N6)(DK/GZ/S4184/17)(a clade 2.3.4.4 d virus), A/chicken/Liaoning/SD007/2017(H5 N1)(CK/LN/SD007/17)(a clade 2.3.2.1 d virus), and A/chicken/Guangxi/SD098/2017(H7 N9)(CK/GX/SD098/17), respectively. The protective efficacy of this novel vaccine and that of the recently used H5/H7 bivalent inactivated vaccine against different H5 and H7 N9 viruses was evaluated in chickens. We found that the H5/H7 bivalent vaccine provided solid protection against the H7 N9 virus CK/GX/SD098/17, but only 50–60% protection against different H5 viruses. In contrast, the novel H5/H7 trivalent vaccine provided complete protection against the H5 and H7 viruses tested. Our study underscores the importance of timely updating of vaccines for avian influenza control.     

Zerumbone attenuates MPP+-induced cytotoxicity in human neuroblastoma SH-SY5Y cells by inhibition of oxidative stress

AIM: To investigate the role of zerumbone (ZER) in 1-methyl-4-phenylpyridinium (MPP⁺)-induced cytotoxicity of human neuroblastoma SH-SY5Y cells. METHODS: Human neuroblastoma SH-SY5Y cells were cultured in vitro and the protective effect of ZER against MPP⁺-induced cytotoxicity was measured by CCK-8 assay. Flow cytometry was used to determine the apoptosis and reactive oxygen species (ROS). The expression of Parkinson disease protein 7 (PARK7) was knocked-down by using PARK7-specific short hairpin RNA (shRNA). The protein levels of PARK7, nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) were determined by Western blot. RESULTS: MMP⁺ remarkably reduced the cell viability in a dose-dependent and time-dependent manner. The SH-SY5Y cell injury model was established by treatment with MPP⁺ at 600 μmol/L for 24 h. ZER up-regulated the protein levels of PARK7 and Nrf2 (P<0.05), alleviated apoptosis (P<0.05), and reduced ROS production (P<0.05) in the SH-SY5Y cell injury model. Meanwhile, N-acetyl-L-cysteine (NAC) had the similar functions. Moreover, significant reductions in the protein levels of Nrf2 and HO-1 (P<0.05), and obvious increases in apoptosis (P<0.05) and ROS level (P<0.05) were demonstrated in PARK7-knockdown cells. CONCLUSION: ZER protects SH-SY5Y cells against MPP⁺-induced cytotoxi-city, which may be related to activation of PARK7/Nrf2/HO-1 pathway, and subsequent attenuation of oxidative stress and apoptosis.     

大花君子兰叶绿体基因组及其特征

采用Illumina MiSeq测序平台对大花君子兰（Clivia miniata）叶片总DNA进行测序，通过组装获得了其叶绿体基因组（cpDNA）全长序列（158 114 bp）。对其cpDNA注释得到135个基因，包含87个蛋白编码基因、40个tRNA基因和8个rRNA基因。采用生物信息学方法对获得的cpDNA进行简单序列重复（SSR）分析和密码子偏好性分析。结果显示：①大花君子兰cpDNA中共有61个SSR位点，其中单核苷酸、二核苷酸、三核苷酸、四核苷酸、五核苷酸和六核苷酸重复数分别为38、9、2、8、3和1个，多数SSR分布在基因间隔区；②大花君子兰cpDNA密码子偏爱以A或U（T）结尾，亮氨酸使用频率最高，半胱氨酸使用频率最低。基于24种植物的cpDNA全长和23种植物的叶绿体ycf2基因序列进行系统发育分析，结果显示大花君子兰与石蒜科植物在同一分支，显示最近的亲缘关系，支持大花君子兰属于石蒜科。基于叶绿体ycf2的系统发育分析结果与基于cpDNA全长的系统发育分析研究结果大部分相同，支持ycf2基因可以代替cpDNA全长用于植物系统发育分析。