通过优化gRNA设计提高CRISPR/Cas9系统中基因编辑效率的方法

CRISPR/Cas9系统是一种广泛应用于动物、植物、真菌、细菌的新一代基因定点编辑技术,如何提高g RNA的编辑效率是该技术的关键点也是许多研究者关注的焦点。本研究利用CRISPR/Cas9技术对番茄miR167a前体序列进行基因编辑,根据番茄miR167a前体序列在线设计候选g RNA,同时结合体外编辑检测的方法筛选出能够在体外成功编辑的g RNA-G1,把其连接到含有荧光标记的pP1C.1C表达载体,转化番茄子叶,产生愈伤组织数116个,得到5个含有表达载体的材料,经测序发现,其中3个材料发生了编辑。结果表明,通过体外检测和在线软件设计相结合的方法来快速检测g RNA的编辑效率,能筛选出最优的g RNA用于载体的构建,与未采用体外编辑检测来设计g RNA的方法相比,显著提高了CRISPR/Cas9的编辑效率。本研究通过优化g RNA,创新了一种高效利用CRISPR/Cas9进行基因编辑的方法。     

Relationship Between Leaf C/N Ratio and Insecticidal Protein Expression in Bt Cotton as Affected by High Temperature and N Rate

Expression of insecticidal protein for transgenic Bacillus thuringiensis （Bt） cotton is unstable and related to nitrogen metabolism. The objective of this study was to investigate the relationship between leaf carbon nitrogen ratio （C/N） and insecticidal efficacy of two Bt cotton cultivars. C/N ratio and Bt protein content were both measured at peak square period and peak boll period respectively under 5-7 d high temperature and different nitrogen fertilizer rates on the Yangzhou University Farm and the Ludong Cotton Farm, China. All plants were grown in field. The results showed that the C/N ratio enhanced slightly and the Bt protein content remained stable at peak square period, but significant increases for the C/N ratio and decreases markedly for the leaf Bt protein concentration were detected at the peak boll period. The similar patterns at the two growth periods were found for the leaf C/N ratio and Bt protein content by different N fertilizer treatments. When nitrogen rate was from 0 to 600 kg ha-l, the C/N ratio was reduced by 0.017 and 0.006 for Sikang 1 and Sikang 3 at peak square period, compared to the 1.350 to 1.143 reduction for Sikang 1 and Sikang 3 at peak boll period, respectively. Correspondingly, the leaf Bt protein contents were bolstered by 2.6-11.8 and 26.9-36.9% at the two different growth periods, respectively. The results suggested that enhanced C/N ratio by high temperature and nitrogen application may result in the reduction of inseetiocidal efficacy in Bt cotton, especially in peak boll period.     

小麦/玉米垄沟套作条件下施肥部位对土壤水氮分布的影响

通过旱棚小车模拟小麦/玉米垄沟套作种植试验,研究了3种不同垄沟部位(垄顶、垄底和沟中)施肥对小麦/玉米生长和产量及土壤水氮分布的影响。结果表明:相同灌水条件下,施肥部位对垄沟套作水分分布影响不显著,但土壤硝态氮的分布差异较大;垄顶施肥和垄底施肥处理下,由于水肥异区,其垄上小麦生长带的硝态氮主要分布在0~30 cm土层,有效地减少了硝态氮向土壤深层淋溶,而后期沟内玉米生长带的水氮处理相同,因此其硝态氮分布差异较小;在相同灌水条件下,垄沟套作可以有效降低水分流失,减少氮肥损失,因此较传统平作更有利于作物生长,提高小麦、玉米的产量,且以垄顶施肥和垄底施肥效果最显著,小麦产量较平作分别增加11.47%、10.81%,玉米产量较平作分别增加18.87%、22.70%。     

基于可见/近红外反射光谱的梨树叶片钾含量的快速测定研究

利用可见/近红外反射光谱定量分析技术对梨树鲜叶钾素含量进行快速测定研究。对150个梨树叶片样本进行光谱扫描,其中120个做建模集,30个做验证集。通过对样品的可见/近红外光谱进行多种预处理,并建立钾素预测模型,探讨了可见/近红外光谱数据预处理对预测精度的影响。结果表明,通过原始光谱与S-G(3)平滑相结合的预处理方法,用17个主成分建立的偏最小二乘法模型最好,其交叉验证集和预测集模型的决定系数(R2)分别为0.722 7和0.679 1,交叉验证均方根误差(RMSECV)为1.171,预测的平均相对误差为6.81%,能高效、快速地预测梨树叶片钾素含量,为梨树钾素快速测定提供了新的手段。     

Effects of Momordica grosvenori Saponins on Type 2 Diabetes Rats Induced by High-sucrose/High-fat Diet and Streptozotocin

The purpose of the experiment was to study the effect of Momordica grosvenori saponins on improving the effect of high-sucrose/high-fat diet combined with streptozotocin induced type 2 diabetes in rats.The type 2 diabetes rat model was established with a high-sucrose and high-fat diet combined with streptozotocin.Successfully modeled rats were given the total saponins of Momordica grosvenori (100,200,400 mg/kg) or metformin (268 mg/kg) by gavage,once a day for 4 weeks.Recording the "three more and one less"index (24 h internal urine output,water intake and feed intake,body weight);Detecting of biochemical indicators,analysis of fasting blood glucose (FBG),serum total cholesterol (TC),triglycerides (TG),low-density lipoprotein cholesterol (LDL-C),high-density lipoprotein cholesterol (HDL-C),insulin (INS),catalase (CAT),superoxide dismutase (SOD),malondialdehyde (MDA),alanine aminotransferase (AST),aspartate aminotransferase (ALT),blood urea nitrogen (BUN) and creatinine (Cr) levels.Hematoxylin-eosin staining (HE) was used to observe the pathological changes of the pancreas.Compared with the model group,the feed intake,water intake,urine output,blood indicators FBG,INS,MDA,TC,TG,BUN and Cr levels,AST and ALT activities were significantly or extremely significantly decreased in the total saponins of Momordica grosvenori each dose group and metformin group (P<0.05;P<0.01),while CAT,SOD activities and HDL-C level were significantly or extremely significantly increased (P<0.05;P<0.01).In pancreatic tissue,the number of pancreatic cells was increased significantly,and the pathological damage of the pancreas was significantly reduced. Momordica grosvenori saponins and metformin had obvious effects on improving glucose metabolism disorders,and at the same time could significantly reduce insulin resistance,enhance the body’s ability to resist oxidative stress and improve liver and kidney functions.     

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。     

潮土小麦碳氮含量对长期不同施肥模式的响应

【目的】以潮土21年长期定位试验为基础,分析不同施肥模式下冬小麦不同生育期的地上部生物量、碳氮含量、碳氮比及碳氮积累量,探讨冬小麦碳氮含量对不同施肥模式的响应规律。【方法】试验包括不施肥(CK)、单施氮肥(N)、施氮磷肥(NP)、施氮钾肥(NK)、氮磷钾配施(NPK)、氮磷钾肥配施有机肥(NPKM)、施氮磷钾肥及玉米秸秆还田(NPKS)7个处理。在2011 2012年冬小麦生长季,分别采集越冬、拔节、灌浆、成熟四个生育时期地上部植株样品,利用Euro Vector EA3000型元素分析仪对小麦植株样品的全碳、全氮含量进行测定。【结果】NPK、NPKM和NPKS处理均能显著提高各生育期小麦地上部干重,其中NPKM处理小麦地上部干重在越冬、拔节、灌浆、成熟期分别比CK提高了111%、194%、238%、206%,除越冬期外,等量氮肥条件下,NPK、NPKM和NPKS 3个处理间小麦同一生育期地上部干重无显著差异,说明与氮磷钾配施相比,有机无机配施与秸秆还田这两种措施并不能显著提高小麦地上部生物量;小麦地上部碳含量受不同施肥影响很小,不同生育期小麦地上部碳含量平均值为410 g/kg;小麦成熟期地上部氮含量以N和NK处理最高,分别达到19.4和18.1 g/kg,其中N处理小麦地上部氮含量分别比NPKM和NPKS处理高52%和66%。随着生育期的推移,各处理小麦氮含量逐渐降低,总体表现为越冬期拔节期灌浆期≥成熟期;在整个生育期中各施肥处理碳含量基本保持不变而氮含量呈逐渐下降趋势,这就使得各施肥处理地上部分C/N比随生育期的推移呈逐渐增加趋势;不同施肥下小麦碳积累量差异性和地上部干物质重差异性规律一致,而不同施肥下地上部氮积累量差异性不同于干物质重的差异性,以NP处理最高,达545 kg/hm2,分别比NPKM和NPKS处理高61%和68%。【结论】施肥方式不能显著改变小麦碳含量但能影响氮含量,因此小麦生物量大小决定了其碳的积累量,相应地,C/N比大小则由氮含量决定。氮磷钾配施、有机无机配施及秸秆还田处理下,小麦具有较高的生物量从而具有较高的碳氮积累量,这有利于增加农田系统碳、氮积累,提升土壤碳、氮肥力。     

滩涂围垦农田土壤饱和导水率的影响因素及转换函数研究

确定苏北沿海滩涂围垦农田耕层土壤饱和导水率的影响因素,构建适合该区的土壤转换函数,是研究该区田间土壤水盐运动和盐渍化防控的重要前提。本文在该区典型地块实测土壤饱和导水率和相关土壤基本理化性质,探讨了该区土壤饱和导水率的剖面分布特点,对影响饱和导水率的土壤基本性质进行了主成分分析,并建立了用于该区饱和导水率间接估算的土壤转换函数。结果表明:滩涂围垦农田土壤饱和导水率随剖面深度增加呈表土层高、亚表层低、底土层又升高的趋势,20~40 cm土层饱和导水率最小,介于2.75~6.73 cm·d-1,属低透水强度;土壤容重随剖面深度增加表现出与饱和导水率相反的变化特点。除了容重、孔隙度、质地等物理因素外,土壤肥力、盐分等化学性质也是影响饱和导水率的重要因素;影响滩涂围垦农田土壤饱和导水率的因素可由持水特性、盐碱状况、养分特征和土壤质地4个主成分反映,其累计贡献率达78.17%。在Vereecken转换函数中引入土壤盐分后可提高预测精度,修正函数Vereecken_1是最适合滩涂围垦农区土壤、具有最佳预测精度的转换函数。本文构建的土壤转换函数,可通过较易获得的砂粒、黏粒、容重、盐分和有机质对耕层土壤饱和导水率进行较高精度的预测,其结果可为滩涂盐渍化农区田间尺度土壤饱和导水率间接估算以及水盐运动数值模拟提供支持。     

4种山茶属植物花粉形态的扫描电子显微观察

为了给山茶属植物的研究提供孢粉学资料,验证花粉形态在植物识别与鉴定应用中的可行性,应用扫描电子显微镜对山茶科山茶属4种植物的花粉表面微形态进行了观察和研究。结果表明,4种山茶属植物花粉在形状、大小、P/E值和外壁纹饰等方面均存在明显的种间差异,其花粉外壁纹饰分属不同的类型:金花茶(Camellia nitidissima)网状,凹脉金花茶(Camellia impressinervis)近脑纹状(近蠕虫状纹饰),蒙自连蕊茶(Camellia forrestii)皱波状,贵州连蕊茶(Camellia costi)皱网状,其中,贵州连蕊茶的网脊上密布疣点(疣状突起),可能是一个进化特征;4种植物的P/E值和P值呈明显的正相关(r2=0.827 2),具有较大的P/E值和P值的两种连蕊茶组植物处于较高的进化地位,山茶属不同种植物花粉外壁纹饰和P/E值反映了物种间的差异,也反映出近缘物种间一定的进化和亲缘关系,可为组间分类及进化关系研究提供孢粉学依据。     

Trends in NE Atlantic landings (southern Portugal): identifying the relative importance of fisheries and environmental variables

Time series of commercial landings from the Algarve (southern Portugal) from 1982 to 1999 were analyzed using min/max autocorrelation factor analysis (MAFA) and dynamic factor analysis (DFA). These techniques were used to identify trends and explore the relationships between the response variables (annual landings of 12 species) and explanatory variables [sea surface temperature, rainfall, an upwelling index, Guadiana river (south‐east Portugal) flow, the North Atlantic oscillation, the number of licensed fishing vessels and the number of commercial fishermen]. Landings were more highly correlated with non‐lagged environmental variables and in particular with Guadiana river flow. Both techniques gave coherent results, with the most important trend being a steady decline over time. A DFA model with two explanatory variables (Guadiana river flow and number of fishermen) and three common trends (smoothing functions over time) gave good fits to 10 of the 12 species. Results of other models indicated that river flow is the more important explanatory variable in this model. Changes in the mean flow and discharge regime of the Guadiana river resulting from the construction of the Alqueva dam, completed in 2002, are therefore likely to have a significant and deleterious impact on Algarve fisheries landings.