Characterization of Genomic Integration and Transgene Organization in Six Transgenic Rapeseed Events

To characterize the DNA rearrangement of both the T-DNA region and the genomic insertion site during T-DNA insertion, the Genomewalker strategy was used to isolate the junctions between the inserted DNA and the plant genomic DNA in six rapeseed events as well as the genomic DNA at the sites before integration. During transformation in each of the six events, portions of both the right border（RB） and left border（LB） regions of the T-DNA were deleted, ranging from a 7 nucleotide deletion of the LB repeats in event RF1 to a 207 bp deletion of the LB region in event RF2. For the six events, T-DNA integration resulted in a deletion at the target site spanning less than 100 bp. Sequence analysis indicated that the T-DNA was integrated into the coding region of various native rapeseed genes in events RF1 and RF2. Duplications of the genomic DNA target site were observed in events RF2, RF3 and Topas 19/2. And multimerization of transgenes was found in event Topas 19/2, in which, the T-DNA was integrated as a head-to-head（RB-to-RB） concatemer into the recipient genome. In event MS1, chromosomal translocation or a large target-site deletion may have occurred during T-DNA integration, which was identified due to a failure to amplify the presumptive insertion site based on the flanking rapeseed DNA sequences. Our results provide comprehensive data concerning transgene organization and the genomic context of the T-DNA in six rapeseed events, which can aid in the developing of insert fingerprinting and the monitoring of long-term genetic stability and potential unintended effects of transgenic events.     

液态有机肥对滴灌棉花光合特性及产量形成规律的影响

为优化滴灌棉花的施肥技术,探究液态有机肥对滴灌棉花生长发育的影响,笔者采取单因子随机区组试验设计,研究在滴灌棉花不同生育时期施用液态有机肥对棉花光和特性、干物质积累及产量的影响。结果表明:(1)施用液态有机肥能够促进植株生长发育,增大光合速率增加棉花产量,且随着液态有机肥施用量的增大,各处理叶面积指数、SPAD值均呈现先增后减的趋势,均于铃期达最大值,其中苗期与蕾期施液态肥的C处理最大值分别为3.25和62.63;(2)叶片净光合速率(Pn)、蒸腾速率(Tr)、气孔导度(Gs)均以C处理最高,并于8月5日花铃期达到极值,而胞间二氧化碳浓度(Ci)表现为C处理最低,为188.67μmol/mol;(3)整个生育期内单株干物质积累量、伏前桃、伏桃、单株铃数及品质均以C处理最好,与其他处理达显著差异水平(P0.05),且增产幅度为27.01%~42.97%。研究表明,液态有机肥适宜的施用时期不仅可提高叶面积指数、叶绿素含量及光合速率,同时为后期干物质积累及产量的形成奠定基础。     

涝渍对Bt棉棉蕾杀虫蛋白表达含量及氮代谢影响

以Bt棉常规种泗抗1号和杂交种泗抗3号为材料,于盛蕾期设计不同程度涝渍(土壤持水量为最大持水量的90%、100%,渍水2 cm)胁迫4 d以及土壤持水量为最大持水量90%逆境下持续胁迫24~120 h的试验,探讨棉蕾中Bt杀虫蛋白表达量变化及相关氮代谢生理机制。结果表明,土壤不同涝渍程度影响棉蕾Bt蛋白含量。土壤持水量为最大持水量的90%、100%和渍水2 cm处理胁迫后,常规种泗抗1号棉蕾中Bt蛋白含量分别下降36.5%、42.1%和51.4%,杂交种泗抗3号分别下降19.2%、57.2%和64.5%。90%的土壤持水量持续胁迫96 h使棉蕾Bt蛋白含量开始显著下降,泗抗1号和泗抗3号分别下降20.8%和17.6%。随着胁迫时间延长下降幅度增大。不同涝渍程度或水渍持续胁迫条件下,棉蕾中可溶性蛋白含量,硝酸还原酶(NR)、谷氨酸草酰乙酸转氨酶(GOT)、谷氨酸丙酮酸转氨酶(GPT)、谷氨酰胺合成酶(GS)和谷氨酸合成酶(GOGAT)活性下降,游离性氨基酸含量,肽酶、蛋白酶活性增加。说明涝渍逆境下,棉蕾中蛋白质合成能力下降、分解能力增强,引起可溶性蛋白包括Bt杀虫蛋白含量下降,导致棉蕾抗虫性下降。     

江汉平原棉花合理施氮量研究

采用田间小区试验研究了江汉平原棉花合理施氮量。结果表明,在施用90 kg/hm~2 P2O5、180 kg/hm~2K2O和3 kg/hm~2持力硼基础上,利用线性+平台模型得到棉花中高产量水平下的合理施氮量为280 kg/hm~2。在不明显减产的条件下,从提高氮肥当季利用率、尽量降低氮肥投入的角度,可以将氮肥用量降低到240kg/hm~2左右,在此施氮水平下仍然可能通过改进田间管理措施获得高产。因此,江汉平原棉花氮肥减量空间为20~60 kg/hm~2。     

不同施磷方式对海岛棉农艺性状及产量的影响

在新疆生态条件下,采用海岛棉新海35号研究同等用量3种施磷方式(犁地前全部撒施、犁地前撒施50%与作种肥施50%和全部作种肥施)对棉花农艺性状及产量的影响。结果表明:施磷方式对棉花生育进程的影响较小,各处理对新海35号棉花的农艺性状没有显著性的影响,P1处理可略为提高棉花花铃期叶面积指数,其中P1处理会略提高棉花后期的干物质积累量,以P1、P2处理的方式施磷肥,棉花的单株结铃数可提高4%~7%皮棉产量可提高12%~14.8%。     

四唑虫酰胺对棉田斜纹夜蛾的防治效果

【目的】研究双酰胺类杀虫剂四唑虫酰胺对斜纹夜蛾的室内生测和田间药效试验。【方法】采用人工饲料混药法测定四唑虫酰胺对斜纹夜蛾3龄幼虫毒力,采用机率值分析方法对数据进行处理,用SAS统计分析系统计算毒力回归方程和LD₅₀。用喷洒药剂悬浮液的方法进行田间试验,使用邓肯新复极差法(DMRT)对不同处理防治效果进行统计分析。【结果】四唑虫酰胺对斜纹夜蛾的LD₅₀为0.58 μg/g,氯虫苯甲酰胺对斜纹夜蛾的LD₅₀为1.12 μg/g,四唑虫酰胺的杀虫活性较高,但与氯虫苯甲酰胺的差异不显著。药后3 d,18%四唑虫酰胺悬浮剂7.5和10 mL/667m²处理对斜纹夜蛾的防效分别为85.00%和87.87%,显著高于5.0 mL/667m² 药剂处理,与5%氯虫苯甲酰胺悬浮剂 45 mL/667m²处理差异不显著。药后7 d,18%四唑虫酰胺悬浮剂 3个浓度处理对斜纹夜蛾的防效与5%氯虫苯甲酰胺 45 mL/667m²处理差异不显著。药后 14 d,18%四唑虫酰胺悬浮剂10 mL/667m²处理的防效显著高于5.0和7.5 mL/667m²药剂处理,但与5%氯虫苯甲酰胺 45 mL/667m²处理差异不显著。【结论】18%四唑虫酰胺悬浮剂对斜纹夜蛾具有良好的的防治效果,且随着浓度增高其防效增大。     

冠县棉花花铃期田间管理技术

阐述了冠县棉花花铃期的生育特点,结合生产实际制定了田间管理关键技术措施,包括重施花铃肥、补施盖顶肥、中耕培土、适时整枝、化学调控、病虫害防治等,以供参考。     

Development and Application of Cotton InDel Markers Based on High Throughput Sequencing

[Objective] Dimorphic InDel markers can be used for cotton variety identification and purity detection, to improve the accuracy and efficiency of cotton seed testing, and to play a role in molecular breeding of cotton. [Method] Based on the whole genome sequencing of 121 cotton varieties from different sources, the InDel markers with high polymorphism were developed according to polymorphism information content(PIC) and were applied in the genetic distance analysis and cluster analysis by using 66 cotton varieties in China. [Result] Totally 10 967 InDel were identified based on the next generation sequencing data of 121 cotton varieties. Among the 85 pairs of InDel primers synthesized, 64 were selected including 35 from At group and 29 from Dt group. The minimum average allelic frequency(MAF) of At and Dt chromosomes were 0.45 and 0.32, respectively, while the PIC were 0.49 and 0.40, respectively. The genetic distances of the 66 cotton varieties ranged from 0.04 to 0.65 centimorgan (cM), with an average of 0.39 cM. The two varieties with the largest genetic distance were Simian 3 and CCRI 36, and the two varieties with the smallest genetic distance were Xumian 18 and Xuza 3. [Conclusion] The 64 cotton dimorphic InDel markers can effectively reveal the relationships among varieties based on the genetic distance, and distinguish cotton varieties from different sources, which has certain theoretical significance and application value.     

花龄期棉花虫害的电子鼻检测

棉花害虫具有隐蔽性、迁飞性和突发性特点，并且影响因素众多，棉花虫害准确地诊断是农业领域的难点问题。该研究以受到棉铃虫侵害的花铃期棉花为研究对象，采用电子鼻对不同处理的棉花挥发物进行检测。研究表明，主成分分析（Principal Component Analysis, PCA）和聚类分析结果显示健康棉花释放的挥发物具有明显的昼夜节律性，健康棉花与虫害棉花差异性显著。径向基函数神经网络（Radial Basis Function Neural Network, RBFNN）对8个不同时间的4组虫害棉花处理进行分析，测试集判别总的正确率为73.4%，健康棉花对照组测试集判别正确率100%，误判样本出现在3个虫害处理之间。当不考虑时间因素建立虫害棉花统一的预测模型，RBFNN模型对健康棉花对照组的预测正确率均达到了100%，分析结果可以作为花铃期棉花是否遭受棉铃虫侵害的依据，说明电子鼻可以作为棉花虫害发生的有效监测手段，在农作物虫害监测领域具有潜在的应用价值。     

中国野生毛葡萄芪合酶基因VqSTS11和VqSTS23调控抗白粉病的研究

利用同源克隆法得到抗白粉病的中国野生毛葡萄‘丹凤–2’芪合酶基因VqSTS11和VqSTS23的开放阅读框，并构建过表达载体；通过器官发生途径诱导不抗白粉病的欧洲葡萄‘无核白’分生愈伤组织并采用农杆菌介导法对其进行转化；经过PCR检测和Western blot鉴定，获得了5株VqSTS11超量表达植株和3株VqSTS23超量表达植株；人工接种白粉菌发现，转基因植株中白粉菌菌丝生长速度慢，孢子萌发受到一定的抑制，并且转基因植株中芪合酶基因相对表达量升高，抗病相关基因表达上调，芪类物质含量积累增多。本研究结果进一步证明中国野生毛葡萄‘丹凤–2’芪合酶基因VqSTS11和VqSTS23对白粉菌具有抗性，‘丹凤–2’可以为改良欧洲葡萄品种抗病性提供抗病基因，作为抗病育种的资源。