Agrobacterium-mediated transformation of the CrDAT gene and selection of transgenic periwinkle lines with a high vincristine accumulation

Catharanthus roseus contains vincristine and vinblastine, which are outstanding drugs for cancer. In the biosynthetic pathways of terpenoid indole alkaloids (TIAs) in C. roseus, deacetylvindoline 4-O-acetyltransferase (DAT) is a key enzyme that catalyses the last reaction of vindoline biosynthesis to form vinblastine and vincristine. In this study, the CrDAT transgene was transferred into the periwinkle by Agrobacterium-mediated transformation and generated transgenic periwinkle lines with an increase in vincristine accumulation. The C. roseus DAT gene was introduced into C. roseus plants and it was confirmed that CrDAT was successfully transferred into the genome of periwinkle plants and efficiently translated to synthesise recombinant DAT protein. Four transgenic periwinkle lines in T1 generation, T1-1, T1-3, T1-6, and T1-7, expressed recombinant DAT protein with the total protein content in the range of 2.86 μg.mg^?1 to 5.12 μg.mg^?1. Moreover, the vincristine contents of four transgenic lines increased by 1.63?2.48-fold compared to non-transgenic plants, ranging from 6.91 µg.g^?1 (fresh weight) to 10.53 µg.g^?1 (fresh weight). The T1-1 line had the highest vincristine content. Hence, the overexpression of the recombinant DAT protein can improve the vincristine accumulation of transgenic C. roseus plants.

Abbreviation: CrDAT - Catharanthus roseus Deacetylvindoline-4-O-Acetyl Transferase; D4H - Deacetoxyvindoline 4-hydroxylase; ELISA - Enzyme-Linked Immunosorbent Assay Monoterpene indole alkaloid; T0, T1 - Generations of transgenic plants; TIAs - Terpenoid indole alkaloids; WT- The wild-type tobacco plants (non transgenic plant); 35S - Cauliflower mosaic virus 35S promoter     

爱媛28’香橙砧与枳砧在南平市引种表现与评价

2017年引进早熟杂柑品种‘爱媛28’香橙砧和枳砧苗分别在大棚设施和露天进行试种，栽培4年调查比较两个砧木组合的生长表现、丰产性与果实品质，结果表明：‘爱媛28’香橙砧树表现生长势强，冠幅、树高、茎粗、结果枝、秋梢等生长量指标显著大于枳砧树，3a树初产1436 kg/667㎡，4a树2523kg/667㎡，显著高于枳砧4a树初产1809kg/667㎡；大棚种植香橙砧4a树果实10月下旬TSS11-12%，12月中旬TSS可达15%，与枳砧相当，可滴定酸比枳砧略高，两者固酸比均达18以上，口感风味极佳，两者单果重、果形指数、果皮厚度、可食率无显著性差异；未密封大棚4a树体经受2020年寒冬极端低温考验无明显冻害，抗旱抗寒能力中等，易感黑点病和流胶病，大棚种植能很好预防黑点病。综合评价爱媛28枳砧和香橙砧均适合在南平市种植，香橙砧比枳砧早期丰产性稳产性好，收益更高，香橙砧‘爱媛28’设施种植较枳砧更具优势。     

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。     

γ⁃戊内酯的制备及其在纤维素生物质转化方面的应用

γ-戊内酯是以木质纤维素生物质为原料制备的一种潜力巨大的平台化合物,它既可转化为高密度燃料、相关高分子材料以及其他高价值化学品,也可作为绿色溶剂促进木质生物质向其他高值方向转化。在化石能源日益紧俏、环境问题日益严重的今天,对γ-戊内酯进行深入研究显得尤为重要。但在实际生产中,仍存在产量低、除杂难等经济环保类问题需要解决。基于γ-戊内酯研究的最新进展,从γ-戊内酯的制备与应用两方面进行了论述,综述了生物质催化生产γ-戊内酯的研究进展,说明不同底物生产γ-戊内酯的理论基础与优缺点,并以贵金属和非贵金属催化剂为界,分类讨论了多种用于合成γ-戊内酯的催化剂。最后,结合γ-戊内酯在纤维素生物质转化应用方面的进展情况,探索了γ-戊内酯与其他相关有机物之间的制备关系,为γ-戊内酯的进一步开发利用提供了思路。     

Notch1 promotes renal tubulointerstitial fibrosis in renal tissues of diabetic mice by inhibiting autophagy through Akt/mTOR signaling pathway

AIM: To observe the changes of Notch1 expression and autophagy in the renal tissues of diabetic mice, and to explore the regulatory effect of Notch1 on tubulointerstitial fibrosis by inhibiting autophagy in diabetic nephro-pathy. METHODS: The mice were randomly divided into normal control group (db/m mice) and diabetes group (db/db mice), with 8 rats in each group. After 12 weeks of feeding, the mice were sacrificed and the corresponding biochemical indexes were measured. The protein expression of Notch1 in the renal tubular epithelial cells was observed by immunohistochemical staining. The protein levels of Notch1, PTEN, p-Akt (Thr308), Akt, p-mTOR (Ser2448), mTOR, LC3, P62, collagen type Ⅰ (Col-Ⅰ) and collagen type Ⅲ (Col-Ⅲ) were determined by Western blot. RESULTS: Compared with the db/m mice, the blood glucose, glycosylated hemoglobin, serum creatinine, triglyceride and total cholesterol were increased in the db/db mice (P<0.01). Renal tubular epithelial cell vacuolar degeneration, renal tubular expansion and interstitial inflammatory cell infiltration in db/db mouse renal tissues with HE staining were observed. The images of Masson staining showed collagenous fiber-like substance deposition in the glomerular capillaries and renal interstitium, and disarrangement of tubular structure in the renal tissues of db/db mice. The protein expression levels of PTEN and LC3-Ⅱ were decreased (P<0.01 or P<0.05), while the protein levels of Notch1, P62, p-mTOR (Ser2448), p-Akt (Thr308), Col-I and Col-III were increased in the db/db mice as compared with the db/m mice (P<0.01). However, no significant change of total mTOR and Akt proteins between the 2 groups was found. CONCLUSION: Notch1 protein expression was increased, PTEN expression was significantly reduced, Akt/mTOR pathway was activated, autophagy was inhibited, and fibrosis was aggravated in the renal tissues of the diabetic mice.     

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.     

中国2种橡胶树炭疽病菌对咪鲜胺敏感性比较

炭疽病是橡胶树上一种重要的叶部病害,主要病原有胶孢炭疽菌和尖孢炭疽菌。为了解中国各植胶区炭疽菌对咪鲜胺的敏感性,采用生长速率法室内测定来自海南、云南、广东和广西4省(区)22株橡胶树胶孢炭疽病菌和15株尖孢炭疽菌对咪鲜胺的敏感性。结果表明:供试的37株橡胶炭疽病菌菌株对咪鲜胺的EC_(50)值为0.022 0~0.272 1 mg/L,平均为(0.061 7±0.055 4)mg/L,EC_(50)最大值是最小值的12.5倍。其中89.2%参试菌株的EC_(50)值介于0~0.100 0 mg/L。根据成组t检验分析结果表明,目前中国4个植胶区的2种橡胶炭疽病菌对咪鲜胺敏感性无明显差异。除了来自海南和云南地区的尖孢炭疽菌有显著性差异外,其他来自不同省份的胶孢炭疽菌和尖孢炭疽菌对咪鲜胺敏感性均无明显差异。研究显示中国橡胶炭疽菌对咪鲜胺仍具有较高的敏感性,但菌株间敏感性有一定的差异,存在一定的潜在抗性风险。     

Cepharanthine induces autophagy via PI3K/AKT/mTOR signaling pathway in ovarian cancer SKOV3 cells

AIM: To investigate the autophagy of human ovarian cancer SKOV3 cells induced by cepharanthine and to explore its mechanism. METHODS: The effect of cepharanthine on the viability of ovarian cancer SKOV3 cells was measured by CCK-8 assay. The SKOV3 cells were treated with cepharanthine, and then the formation of autophagosome was observed with acridine orange staining under fluorescence microscope. The protein levels of LC3, AKT, p-AKT, mTOR, p-mTOR and GAPDH in the SKOV3 cells treated with cepharanthine were determined by Western blot.RESULTS: Cepharanthine significantly inhibited the viability of ovarian cancer SKOV3 cells in a dose-dependent manner (P<0.05). The number of the intracellular acidic autophagosomes with bright red fluorescence was significantly increased after cepharanthine treatment in the SKOV3 cells. The expression of LC3-Ⅱ in SKOV3 cells was significantly enhanced after cepharanthine treatment. Furthermore, treatment with cepharanthine in the SKOV3 cells also resulted in a significant down-regulation of phosphorylated form of AKT and mTOR (P<0.01), while the total protein level was not changed. Combination of cepharanthine and 3-methyladenine resulted in a substantial decrease in the cell viability compared with using cepharanthine alone.CONCLUSION: Cepharanthine significantly inhibits the growth of human ovarian cancer SKOV3 cells and induces the autophagy, which may be correlated with down-regulation of PI3K/AKT/mTOR signaling pathway.