大豆PAL2-3基因的克隆及转化研究

大豆异黄酮是苯丙氨酸代谢途径中合成的一类次级代谢产物,在苯丙氨酸代谢途径中,苯丙氨酸解氨酶(PAL)是关键酶和限速酶。本课题组前期研究发现PAL基因的相对表达量与大豆异黄酮含量具有明显的协同增减趋势,并且在PAL基因家族成员时空表达模式分析中发现,PAL2-3(XM_003542493)是PAL基因家族中相对表达量较高的主要表达成员之一。本试验首先通过克隆大豆中PAL2-3基因;然后构建pCAMBIA3301-GmPAL2-3植物过表达载体,将构建好的pCAMBIA3301-GmPAL2-3表达载体重组质粒转化到根癌农杆菌EHA105中;采用农杆菌介导的大豆子叶节转化体系获得转化植株,并对T1代转化植株进行PPT检测,外源标记基因Bar检测以及荧光定量PCR检测,对转基因阳性植株进行大豆籽粒异黄酮含量的测定。结果表明T_1代转基因植株中PAL2-3基因的表达量是对照的5.11~11.24倍,总异黄酮含量最高的(2 587.63μg·g~(-1))是对照(1 616.90μg·g~(-1))的1.6倍。因此在大豆中过量表达PAL2-3基因可以提高大豆籽粒中异黄酮含量。     

脲酶抑制剂与硝化抑制剂对稻田土壤氮素转化的影响

【目的】本研究旨在阐明脲酶抑制剂(urease inhibitor,UI)和硝化抑制剂(nitrification inhibitor,NI)对稻田土壤氮素转化的影响,探讨抑制剂提高稻谷产量以及氮肥利用率的机理。【方法】本试验设在我国南方红壤稻田,共5个处理:1)不施氮肥(CK);2)尿素(U);3)尿素+脲酶抑制剂(U+UI);4)尿素+硝化抑制剂(U+NI);5)尿素+脲酶抑制剂+硝化抑制剂(U+UI+NI);脲酶抑制剂采用N-丁基硫代磷酰三胺(NBPT),硝化抑制剂采用3,4-二甲基吡唑磷酸盐(DMPP)。在水稻分蘖期和孕穗期测定土壤脲酶活性、硝酸还原酶活性、土壤铵态氮含量、硝态氮含量以及微生物碳、氮的含量,分析NBPT与DMPP对水稻两个主要生育期土壤氮素供应的影响,比较各处理的产量以及氮肥利用率,通过逐步回归分析研究以上各指标对产量的影响,探明脲酶抑制剂NBPT与硝化抑制剂DMPP在稻田的增效机理。【结果】1)与单施尿素相比,添加NBPT以及NBPT与DMPP配施均显著提高稻谷产量与地上部氮素回收率,两个处理分别增产6.56%与8.24%,氮素回收率提高幅度为19.4%与23.7%。2)与单施尿素相比,添加NBPT以及NBPT与DMPP配施,显著降低水稻分蘖期的土壤脲酶活性和铵态氮含量,显著提高孕穗期的铵态氮含量,而对此时期的脲酶活性无显著影响,所有处理对两个时期的硝态氮含量、硝酸还原酶活性、微生物量碳、氮含量均无显著影响;因此,NBPT对于抑制脲酶活性以及提高铵态氮含量的作用主要在孕穗期之前,而单施DMPP没有显著效应。3)从各项土壤指标与水稻产量相关性的逐步回归分析结果来看,水稻分蘖期与孕穗期稻田土壤中铵态氮含量对水稻产量影响显著,而且孕穗期的影响大于分蘖期,其余指标则对产量无显著影响。【结论】脲酶抑制剂NBPT以及NBPT与硝化抑制剂DMPP配施显著提高孕穗期土壤中的铵态氮含量,显著提高稻谷产量以及地上部氮素回收率,证明了生产上氮肥后移的重要意义。     

Transgenic sorghum with suppressed synthesis of kafirin subclasses: Effects on flour and dough rheological characteristics

Arising from work showing that conventionally bred high protein digestibility sorghum types have improved flour and dough functionality, the flour and dough properties of transgenic biofortified sorghum lines with increased protein digestibility and high lysine content (TG-HD) resulting from suppressed synthesis of several kafirin subclasses, especially the cysteine-rich γ-kafirin, were studied. TG-HD sorghums had higher flour water solubility at 30 °C (p < 0.05) and much higher paste viscosity (41% higher) than their null controls (NC). TG-HD doughs were twice as strong as their NC and dynamic rheological analysis indicated that the TG doughs were somewhat more elastic up to 90 °C. CLSM of doughs and pastes indicated that TG-HD had a less compact endosperm protein matrix surround the starch compared to their NC. The improved flour and dough functional properties of the TG-HD sorghums seem to be caused by reduced endosperm compactness resulting from suppression of synthesis of several kafirin subclasses which modifies protein body and protein matrix structure, and to improved protein-starch interaction through hydrogen bonding specifically caused by reduction in the level of the hydrophobic γ-kafirin. The improved flour functionality of these transgenic biofortified sorghums can increase their commercial utility by complementing their improved nutritional quality.     

不同施氮水平下缓释氮肥配施对机插稻氮素利用特征及产量的影响

【目的】在高施氮水平和常规施氮水平下,研究缓释氮肥配施对机插稻氮素利用和产量的影响及其生理机制,为机插稻的氮肥高效利用提供依据。【方法】以中迟熟杂交籼稻川谷优7329为材料,采用二因素裂区设计,设置不同的施氮水平及5种缓释氮肥与常规氮肥(尿素)配施处理,并设置不施氮处理,分析不同施氮水平下缓释氮肥与常规氮肥配施对机插稻氮素利用和产量的影响及其生理机制,并探讨氮素利用和产量与生理响应之间的关系。【结果】结果表明,施氮水平、缓释氮肥与常规氮肥配施对机插稻氮素利用特征及产量均存在显著或极显著的影响,机插稻拔节、抽穗及结实期对氮素的吸收利用以及结实期茎鞘的氮素转运量与干物质量、氮素积累量、氮肥表观利用率、每穗实粒数及最终产量显著或极显著正相关(r=0.38*~0.69**)。与常规氮肥运筹相比,缓释氮肥与常规氮肥配施、全缓释氮肥施用处理的机插稻成熟期干物质积累量、氮素积累总量、光合势、叶面积指数、氮肥表观利用率及穗部氮素增加量均显著提高,进而促进增产。【结论】据产量及氮素利用效率表现,机插稻产量在180 kg/hm2施氮水平下较高,且在缓释氮肥与常规氮肥配施比例为7∶3一次性施用时产量最高,较其他氮肥运筹产量高0.84%~26.59%,氮肥表观利用率高0.28%~47.02%,为最优氮肥运筹模式。但不同施氮水平下,随着缓释氮肥配施比例的降低,群体叶面积指数、光合势及物质积累等指标也降低,且在施氮处理为全常规氮肥时最低,均不利于机插稻氮肥利用效率及最终产量的提高。     

重组白细胞介素-2高效纯化方法建立

依次通过离子交换法、凝胶过滤法纯化重组白细胞介素-2(IL-2)粗品,利用SDS-PAGE电泳和hIL-2 Elisa kit检测纯化后重组IL-2的纯度和浓度。结果表明,纯化后的重组IL-2纯度和浓度高。本纯化方法简单、稳定、纯化效果好,可为重组IL-2的生产工艺提供技术支持和依据。     

7株根瘤菌菌株与热研2号柱花草共生最适磷浓度研究

采用水培的方法,设3个磷水平,7个菌株,通过对株高、茎叶鲜重、茎叶干重、固氮酶活性、茎叶氮含量、茎叶磷含量的测定及综合分析,确定使热研2号柱花草接种不同根瘤菌菌株达到最佳促生性能的磷浓度。结果表明:7个菌株在中磷时鲜重和含氮量最大,低磷不利于鲜重和含氮量的增加;经主因子综合分析,菌株YM11-1、RJS9-2、PN13-3在高磷时对热研2号柱花草有最好的固氮促生效果,菌株LZ3-2、BS1-1、PN13-3在中等磷条件下有最好的固氮促生效果,菌株FS3-1-1在高磷和中磷都有最好的固氮促生效果。     

肺气虚证小鼠模型造模方法及其对小鼠免疫功能的影响

为筛选肺气虚证小鼠模型最佳造模方法并验证其免疫功能变化，分别选用SO2法、SO2＋寒冷因素刺激法、香烟烟熏法和香烟烟熏＋寒冷因素刺激法重建肺气虚证小鼠模型，检测其体重、肺脏剖检及病理组织变化等指标，筛选最佳造模方法，并测定其腹腔巨噬细胞活性、体外B细胞抗体分泌能力、血清NO含量、淋巴细胞增殖能力及细胞因子IL-2、IL-4、IL-6、IL-10、IFN-γ、TNF-α含量、肺匀浆中sIgA、sIgM水平。结果显示，香烟烟熏＋寒冷因素刺激法小鼠造模完成后出现严重的肺出血、肺组织坏死及炎性细胞浸润等病变，且与对照组小鼠相比，其体重、脾脏指数、胸腺指数分别显著下降18．35％，37．12％，19．69％，肺脏指数显著升高12．73％；小鼠腹腔巨噬细胞活性（P＜0．01）、血清NO水平下降（P＜0．05）；T、B淋巴细胞增殖能力显著升高；细胞因子IL-2（P＜0．01）、IFN-γ、IL-4含量升高（P＜0．05），IL-6含量下降（P＜0．05）；sIgA、sIgM水平均极显著升高。表明，香烟烟熏＋寒冷因素刺激法是肺气虚小鼠模型最佳造模方法，且其免疫功能受损。     

Rate-specific responses of prokaryotic diversity and structure to nitrogen deposition in the Leymus chinensis steppe

Serious nitrogen (N) deposition in terrestrial ecosystems causes soil acidification and changes the structure and function of the microbial community. However, it is unclear how these changes are dependent on N deposition rates, other factors induced by N (e.g., pH), and their interactions. In this study, we investigated the responses of soil prokaryotic community structure and stability after a 13-year N addition in the semi-arid Leymus chinensis steppe in Inner Mongolia, China. Our results demonstrated that the prokaryotic community structure changed at the low N addition rate of 1.75 g N m⁻² yr⁻¹; however, dramatic changes in microbial abundance, respiratory quotient, and prokaryotic diversity occurred at N addition rates of more than 5.25 g N m⁻² yr⁻¹ when the soil pH dropped below 6.0. The two patterns indicated the difference in driving forces for different microbial properties. The N-driven and pH-driven processes are likely the most important mechanisms determining the responses of bacterial community to N. Some copiotrophic/oligotrophic bacteria, e.g., Proteobacteria and Acidobacteria, changed their relative abundances with the N addition continuously even at a low rate, indicating that they were more sensitive to N directly. Some bacterial groups significantly changed their relative abundance at a high N addition rate when pH dropped below 6.0, e.g., Verrucomicrobia and Armatimonadetes, indicating that they were more sensitive to pH below 6.0. N addition altered the prokaryotic community structure through enrichment of copiotrophic bacteria (species adjustment) at low N addition rates and through enrichment of nitrophilous taxa and significant loss of diversity at high N rates. The results also demonstrated that a high N addition diminished the stability of the prokaryotic community structure and activity through reduction in species diversity and bacterial interaction. Overall, this study supported the hypothesis that the responses of prokaryota to N were dependent on deposition rates, and N-driven and pH-driven processes were the important mechanisms to control the shift of the prokaryotic community.     

利用CRISPR/CAS9技术编辑水稻香味基因Badh2

【目的】香稻作为一类特殊的水稻群体,以其清香可口的品质特性备受消费者的欢迎。到目前为止,水稻中的香味主要受第8染色体上编码甜菜碱醛脱氢酶基因Badh2控制。【方法】通过CRISPR/CAS9技术对中花11的香味基因Badh2进行编辑。【结果】获得转基因T_0代植株并对其所衍生的T_1代20个单株进行了鉴定分析,获得了一个剔除了载体骨架且第1外显子上插入一个碱基T的突变体材料。该材料中Badh2 RNA水平显著下调;利用GC-MS技术测定野生型及突变体材料籽粒2-乙酰-1-吡咯啉含量,结果表明突变体材料中的香味物质显著增加;此外,我们还对野生型及T_2代香型植株水稻产量及稻米蒸煮食味品质进行了考查及测定分析,发现除分蘖数及结实率呈现出显著差异外(P0.05),其余各项指标在两组材料间都无显著差异。【结论】通过CRISPR/CAS9技术成功地对水稻香味基因进行了编辑,可为香稻育种提供丰富的理论指导,加快香稻的育种进程。