利用CRISPR/Cas9敲除葡萄VviPDS1基因的研究

选择葡萄八氢番茄红素脱氢酶(Phytoene desaturase)基因VviPDS1为靶标,利用CRISPR/Cas9系统构建基因敲除载体,瞬时转化葡萄叶片原生质体,检测到不同类型的突变。通过农杆菌介导转化‘无核白’葡萄胚性愈伤组织,筛选获得卡那霉素抗性植株71株。经PCR鉴定,其中53株为阳性植株,阳性率为74.64%。测序结果表明,共有20株在靶点发生不同类型的突变,编辑效率为37.74%;其中9株产生了双等位基因突变。对其进行氨基酸序列预测,在第202位氨基酸之后发生了不同程度的变异。利用CRISPR/Cas9系统敲除VviPDS1获得的突变体植株呈现整体矮化,其叶片出现不同程度白化。表明CRISPR/Cas9系统可以通过细胞中的瞬时或稳定表达进行基因编辑,可以实现在葡萄编辑植株中产生纯合敲除。     

Impact of soil water content on maize responses to the plant growth-promoting rhizobacterium Azospirillum lipoferum CRT1

Members of the bacterial genus Azospirillum are root-associated bacteria that increase yield in cereals by promoting growth and alleviating drought stress. How plants integrate the many bacterium-derived growth-promoting stimuli with other environmental factors to generate a coordinated response remains unresolved. Using a commercial Azospirillum strain, A. lipoferum CRT1 and two host maize cultivars, it was observed that bacterization reduced the drought-induced increase in lateral root growth and enhanced the flood-induced increase in lateral root growth in the more drought- and flood-sensitive cultivar. In the other one, A. lipoferum CRT1 only elicited a moderate root growth response under low soil water potential. The photosynthetic potential and activity were increased in the earlier cultivar and decreased in the later one, irrespective of the soil water content. No impact of the bacterium was seen on the growth of the leaves of both cultivars under both stresses until the third leaf stage, therefore suggesting that it is a consequence of multiple primary adaptations to biotic and abiotic stresses. It is suggested that host–bacteria recognition leads to a stress-specific modulation of the root response and a differential stress-independent effect on photosynthesis. This is the first report of the impact of Azospirillum under flood conditions.     

甘肃、青海地区小麦条锈菌监测及群体遗传多样性分析

【目的】了解甘肃和青海小麦条锈菌（Puccinia striiformis f. sp. tritici）春季流行传播路线、群体遗传多样性和生殖模式,明确春季流行期两省小麦条锈菌的传播关系及菌源交流规律,进而为两省小麦条锈病的预测预报、确定越夏初始菌源来源和有效治理提供理论依据。【方法】选择条锈病常发生的地区作为调查和研究区域。甘肃省4个试验点：陇南市文县、陇东平凉市崆峒区、中部麦区定西市临洮县、临夏州临夏县;青海省2个试验点：西宁市城北区、海东市互助县。2017年秋季,在甘肃和青海省6个试验点内根据当地小麦播种适期依次种植82份变异观察圃材料。2018年4—8月,对试验点82份变异观察圃材料进行田间病害调查,并采集到551份小麦条锈菌标样,使用15对引物进行SSR分子标记分析。利用GenAlEx和POPPR v2.5.0软件对数据进行相关分析, 不显著的rbarD值表示连锁平衡,用于推断群体是否发生有性重组。【结果】82份变异观察圃材料在甘肃地区发病比青海地区严重。15对引物组合共扩增出81个位点,每对引物组合产生的多态性位点为2—12个。551份样本克隆矫正后,共鉴定出505个多位点基因型（MLG）,其中仅有32个MLG被克隆并进行了2—6次重新采样。甘肃和青海群体总的基因型多样性（G=0.917）较高,其中,甘肃平凉群体的最高,青海互助群体次之,甘肃临洮群体最低。小麦条锈菌的遗传变异主要在各群体内部个体之间。春季流行期,菌源在各群体之间交流频繁,青海东部（互助和西宁）群体与甘肃（平凉和临夏）群体之间的基因流高于青海（互助和西宁）群体与甘肃文县群体之间的基因流。最小时空网络图（MSN）和非参数主成分分析（DAPC）表明青海互助和西宁的群体与来自于甘肃平凉和临夏的群体之间菌源关系最密切,差异最小;与临洮群体遗传距离相对较远且临洮群体相对独立;文县群体则是一个完全独立的群体,与其他5个群体之间的差异最大。连锁不平衡分析表明,甘肃文县、临夏和青海西宁群体存在不显著的rbarD值表示连锁平衡,是有性生殖群体,其中文县群体（rbarD=0.0139,P=0.186）显示出明显的有性重组特征。【结论】小麦条锈病春季流行期,甘肃地区与青海东部地区的传播路线以甘肃平凉、临夏到青海的传播为主,甘肃文县到青海的传播为辅。甘肃文县、临夏和青海西宁3个群体存在有性生殖现象,对甘肃、青海地区条锈菌丰富的遗传多样性的形成具有一定作用。     

无机碳源对零价铁介导的自养脱氮序批式反应器处理高氨氮废水的影响

无机碳源作为自养微生物的能量来源,是影响自养脱氮细菌富集的重要因素。文章通过添加不同量的KHCO3作为ZVI介导的自养脱氮体系中无机碳源,研究反应体系的脱氮效果影响趋势以及适宜的无机碳源添加量。结果如下:1)KHCO3作为无机碳源可以显著提高NH^+4-N去除率,KHCO3添加量越多,其NH^+4-N去除率越高。KHCO3添加量分别为2 g,1 g,0.5 g的SBR反应器R2,R1,R0.5的NH^+4-N去除率分别为98.39%,65.18%,44.56%,相较不添加KHCO3的R0分别提高86.5%,53.29%,32.67%。ZVI的添加会降低KHCO3对NH^+4-N氧化的促进作用。2)KHCO3可明显提高TIN去除效果,促进总氮脱除的高低顺序是R1>R0.5>R2,SBR反应器R2,R1,R0.5的平均TIN去除率分别为19.01%,32.04%,27.62%,相较于空白组R0分别提高了10.60%,23.63%,19.21%。3)KHCO3可中和硝化反应产生的H^+,对反应体系具有缓冲作用,可使微生物处于较适宜的酸碱环境中,更有利于反应器的稳定运行。4)适量的无机碳源KHCO3可以促进氨氧化细菌和厌氧氨氧化细菌的活性。该研究探讨了无机碳源在零价铁脱氮体系中的影响趋势,为铁型脱氮技术提供了理论支撑。     

Cf-2/Rcr3pim番茄品系对南方根结线虫的抗性测定

Cf-2/Rcr3~(pim)基因型番茄不仅能够抵御番茄叶霉菌的侵染,而且对马铃薯金线虫的寄生也有一定的抑制效果。为挖掘根结线虫的新抗性资源,本研究采用室内人工接种法测定了Cf-0/Rcr3~(pim)、Cf-2/Rcr3-3和Cf-2/Rcr3~(pim)基因型番茄品系对南方根结线虫的抗感性。抗性评价结果显示,Cf-0/Rcr3~(pim)品系对南方根结线虫表现高感,Cf-2/Rcr3-3品系为中感,而Cf-2/Rcr3~(pim)品系则为感病。与Cf-0/Rcr3~(pim)和Cf-2/Rcr3-3基因型相比,Cf-2/Rcr3~(pim)基因型番茄品系虽然对南方根结线虫侵染的敏感性略低,但是不能阻止线虫在根系上的大量繁殖,不适于根结线虫的防控应用。     

ROCK promotes high glucose-induced cardiomyocyte apoptosis by inhi-biting PI3K/Akt signaling pathway

AIMTo investigate whether Rho-associated coiled-coil kinase (ROCK) is involved in high glucose-induced apoptosis of primary cardiomyocytes by regulating PI3K/Akt signaling pathway. METHODSPrimary Wistar rat cardiomyocytes were cultured and identified by α-sarcomeric actin (α-SCA) immunohistochemistry. Cardiomyocytes were treated with 5.5, 33 and 40 mmol/L glucose for 48 h. The cell viability was measured by MTT assay, and the mRNA expression of ROCK1 and ROCK2 in the cardiomyocytes was detected by RT-qPCR. Flow cytometry was used to analyze the apoptosis of the cardiomyocytes. The protein levels of ROCK1, ROCK2, cleaved caspase-3, Bcl-2, PI3K, Akt and p-Akt were determined by Western blot. In order to confirm the regulatory effect of ROCKs on PI3K/Akt signaling pathway, the cells were divided into control group (5.5 mmol/L glucose), high glucose group (33 mmol/L glucose) and high glucose+Y27632 (ROCK inhibitor) group. Western blot was used to detect the protein levels of ROCK1, ROCK2, PI3K, Akt and p-Akt. RESULTSAfter 48 h of high glucose exposure, the values of relative cell viability in 33 and 40 mmol/L glucose groups were (79.71±2.43)% and (68.41±7.49)%, respectively, both of which were significantly decreased compared with normal control group (P<0.05). After 48 h of high glucose exposure, the relative mRNA levels of ROCK1 and ROCK2 in 33 and 40 mmol/L glucose groups were significantly increased compared with normal control group (P<0.05). Compared with normal control group, the apoptotic rate in 33 and 40 mmol/L glucose groups was increased significantly (P<0.05). Compared with normal control group, the protein expression of ROCK1, ROCK2 and cleaved caspase-3 in 33 and 40 mmol/L glucose groups was increased (P<0.05), while the protein expression of Bcl-2 was decreased (P<0.05). No significant difference in the protein levels of PI3K and Akt among the 3 groups was observed, while the protein level of p-Akt in 33 and 40 mmol/L glucose groups was decreased compared with normal control group (P<0.05). Compared with high glucose group, the expression of ROCK1 and ROCK2 was decreased in high glucose+Y27632 group. No significant difference in the protein levels of PI3K and Akt among the 3 groups was observed. Compared with normal control group, the protein level of p-Akt in high glucose group was decreased, and the protein level of p-Akt in high glucose+Y27632 group was increased significantly compared with high glucose group. CONCLUSION Under high glucose environment, ROCK may reduce the level of p-Akt by inhibiting the PI3K/Akt signaling pathway， thus promoting the apoptosis of cardiomyocytes.     

5味中药醇提物对尼罗罗非鱼白细胞免疫活性的作用

将超微五倍子、白头翁、地榆、柯子、虎杖粉碎后过160目筛,按照料液比1∶10将药粉与60%的乙醇水溶液混合,再用超声波粉碎机超声1 h,用滤纸过滤收集滤液,置于多样品平行蒸发仪中浓缩后制得中药提取物。用吸取0.2 mL抗凝剂的无菌注射器自尼罗罗非鱼尾静脉采血0.5 mL,与L-15细胞培养基按体积比1∶9的比例混合成血浆,与中草药提取液单用、联用共孵育,利用氮蓝四唑还原法和流式细胞仪技术分别测定中草药提取液对尼罗罗非鱼外周血白细胞呼吸爆发活性和白细胞吞噬活性的影响。试验结果显示,五倍子、白头翁、地榆、柯子、虎杖单用均能显著提高白细胞氧呼吸爆发活性,呼吸爆发增加率分别为128.68%、123.96%、28.37%、44.44%和78.93%;试验组白细胞吞噬率分别较对照组增加44.04%、44.66%、95.00%、10.35%和64.93%。在两味中药双联用试验中,对白细胞氧呼吸爆发活性呈协同作用的组合为白头翁+地榆,呼吸爆发联用指数为1.28;对白细胞吞噬活性呈协同作用的组合为白头翁+虎杖、柯子+虎杖、五倍子+虎杖和白头翁+柯子,吞噬联用指数分别为11.45、6.20、3.03和1.06。     

利用CRISPR/Cas9系统敲除葡萄中VviEDR2提高对白粉病的抗性

利用CRISPR/Cas9系统定点编辑葡萄白粉病感病基因VviEDR2（Enhanced disease resistance 2），在VviEDR2的DUF1336结构域设计靶位点VviEDR2-T1，构建CRISPR/Cas9敲除载体，通过农杆菌介导法转化‘无核白’葡萄胚性愈伤组织。对PCR阳性植株进行靶位点扩增测序，结果表明，共有8个转基因植株在靶位点处发生不同类型的双等位基因突变，编辑效率为32%；突变体植株生长势较弱，叶片较小，茎秆丛生、细弱。进一步对突变体植株进行抗病检测，结果表明，接种葡萄白粉菌（Erysiphe necator Schw.）5 d后，突变体植株叶片上白粉菌孢子仅能萌发出少量较短初级菌丝，表皮细胞产生大量明显的H2O2，而野生型叶片中白粉菌萌发出大量初级菌丝、次级菌丝和吸器，无明显H2O2产生。这些结果表明，可以利用CRISPR/Cas9技术编辑葡萄感病基因VviEDR2，提高葡萄白粉菌抗性。     

新型缓控释增效肥研究进展和发展前景

传统肥料面临着利用率低、产能过剩等问题,新型缓控释肥通过新型包膜材料或者纳米技术,实现养分释放速度的可控性;通过添加信号物质,实现植物和化肥的互动,将植物养分需求规律作为主导因素,实现肥料的按需供给,达到减少化肥用量,提高肥料利用效率的目的。从定义、研发背景、分类及发展趋势等方面介绍了新型缓控释肥的研究进展,并对其未来发展进行了展望。     

开式离心纸浆泵叶轮流道内部流场PIV试验研究

为研究开式纸浆泵叶轮流道内纤维颗粒对液相流场的影响,采用透明有机玻璃材料制造模型样机,以清水和0.1%质量浓度的头发纤维悬浮液为介质,通过PIV试验对叶轮流道内的流动进行可视化研究.试验结果表明:当输送清水介质时,在大流量工况下叶轮流道内流动稳定,未出现旋涡和低速区;在小流量工况下,当叶轮流道旋转到蜗壳隔舌附近位置时,在叶轮流道中段压力面附近开始产生低速区,且低速区随着叶轮流道靠近隔舌位置和流量减小逐渐增大,在远离隔舌位置的叶轮流道内未产生明显的低速区;受叶片扭曲的影响,在叶轮流道内垂直于泵轴的不同截面上的相对速度大小不同,且随着叶片扭曲程度减小,相对速度差减小;输送头发纤维悬浮液介质时,在纤维颗粒的影响下,叶轮流道内的液相流动相对速度降低,导致在小流量工况下叶轮流道中段压力面附近低速区面积增大;在大流量工况下,纤维颗粒存在使液相流动的相对速度大小分布更均匀.