大豆基因工程根瘤菌田间结瘤和共生固氮效应

利用三亲本接合转移方法,将快生型大豆根病菌B_(52)的基因文库克隆转移到受体菌慢生型大豆根瘤菌2210中,获得4株大豆基因工程根瘤菌(32.43、B—2—6、B—3—8)。田间接种试验表明:在大豆分枝期(V2),43和32结瘤效分别比不接种对照增加117.9％和100.4％;在初花期(R_1),32和B—2—6结瘤数分别比对照增加122.5％和91.6％,根瘤固氮酶活性增加233.3％和195.6％;在结荚始期(R3),B—2—6和32结瘤数比对照增加78.5％和70.1％,根瘤固氟酶活性43和B—2—6分别比对照增加53.4％和49.3％。产量统计表明:32、B—2—6和43比对照分别增产16.8％、14.3％和12.2％,亩增收大豆分别为28.2、24.1和20.6公斤。以上三个菌株均比受体菌株2210增产率高。     

脲酶抑制剂氢醌对土壤尿素氮转化的影响

本文根据用标记和非标记尿素进行的培养试验,论述了氢醌对于尿素的水解、氨的释出和挥发、硝化和反硝化作用以及生物固持的影响。得出的结论是:氢醌的作用不仅在于延缓尿素的水解和减少随之而来的氨的挥发,更重要的,是影响了尿素水解产物进一步转化的进程,增强了尿素氮对于作物的有效持续供应和减少了它的总损失。本文认为,在脲酶抑制剂的研究中,着眼点当不仅在于它们的直接作用,而更需要涉及对尿素氮转化的一系列过程的影响。这样,才能对抑制剂的作用机理有更深入的了解,对它的作用效果有更全面的评价。     

脲酶抑制剂氢醌的环境效应评价

本文根据用标记和非标记氢醌进行的模拟、盆栽和田间定位试验,结合国内外文献有关氢醌的环境常数,论述了氢醌在土壤-植物系统的去向和代谢途径、对土壤酶活性的影响及其环境效应。得出的结论是:作为脲酶抑制剂使用的微量氢醌(0.3—0.4％,与尿素重量比),不会从土壤中淋失和挥发,在土壤和植物中没有累积,对与碳、氮和磷转化有关的土壤酶活性很少影响。在土壤中,它将通过氧化、臭氧化和生物学降解,经由环断裂生成二元酸或参与腐殖物质的合成。在植物体内,主要通过糖苷化得到同化和利用。因此,氢醌作为脲酶抑制剂在农业生产中应用是安全的。     

双拷贝固氮结瘤基因的大豆根瘤菌R.fredii基因工程菌的耐…

用热处理消除了快生型大豆根瘤菌Ｒ．ｆｒｅｄｉｉＵＳＤＡ１９４的非共生质粒,获得了突变株Ｈｏ４,以Ｈｃ４为受体菌,以Ｅ．ｃｏｌｉＨＢ１０１Ｅｃ８３（ＰＬＡＦＲＩ．ｎｏｄｎｉｆ）为供体菌,Ｅ．ＣｏｌｉＰＲＫ２０１３为助体,进行三亲本杂交,得到双重ｎｏｄｎｉｆ基因拷贝的杂合子Ｈｃ４－８。用它们完成共生固氮结瘤试验,结果表明,在初花期Ｈｃ４的固氮酶活性比出发株ＵＳＤＡ１９搞７５．７％Ｈｃ４－８比ＵＳＤＡ１     

生态条件对马占相思结瘤固氮的影响

本文研究了马占相思（Ａｃａｃｉａｍａｎｇｉｕｍ）结瘤固氮和生态条件的关系．结果显示．马占相思根瘤固氮活性的昼夜变化与固氮能源的供给有关,它受光、温影响较大,固氮活性昼夜变化的范围为１—５μｍｏｌＣ２Ｈ４·ｇ－１ｆｒｅｓｈｎｏｄｕｌｅｈ－１。固氮活性的季节性差异也很明显,且与温、湿度的变化关系密切,在温、湿度较好的５月—１０月．固氮活性较高．为３—１０μｍｏｌＣ２Ｈ４·ｇ－１ｆｒｅｓｈｎｏｄｕｌｅｈ－１．冬春的干旱和低温会影响根瘤的生长和存活．造成固氮活性降低甚至失去活性．不同年份和林地的根瘤生物量为１０４—６２５ｋｇ·ｈａ－１,以幼林期根瘤生物量较高．多数样地的根瘤生物量在３００ｈｇ·ｈａ－１以上．随着森林生态系统的发展．根系往土层深处生长以及林下草本和灌木层的增长等原因,根瘤生物量会受影响而有所下降。施肥松土能提高根瘤生物量５７—３４４ｋｇ·ｈａ－１,对增加固氮量有重大意义。     

大豆结瘤突变体成分和初步分析及其对硝酸还原酶活性和结瘤的影响

超结瘤大豆ｎｔｓ３８２和不结瘤大豆Ｎｏｄ４９的叶和根组织水提取物经Ｓｅｐｈａｄｅｘ Ｇ２５过滤,洗脱,再根据洗脱物对硝酸还原酶活性的影响可划分为４个组玢样品,即ｎｔｓ３８２Ｆ１,ｎｔｓ３８２Ｆ２,ｎｔｓ３８２Ｆ３和ｎｔｓ３８２Ｆ４。其中,ｎｔｓ３８２Ｆ２和Ｆ４抑制ＮＲ活性作用在接种ＵＳＤＡ１１０后明显下降。     

脲酶抑制剂氢醌对土壤脲酶活性,氨挥发和硝化的抑制动态

1.氢醌对土壤脲酶活性的抑制率及其持续的时间同氢醌浓度成正相关,与土壤脲酶活性成负相关。2.氢醌能有效地抑制施入土壤中尿素氨的挥发,而对铵盐和尿素的硝化强度产生强烈抑制。3.在麦秸还田土壤中,由于脲酶活性增高而提高了施入尿素的水解速度,故需提高氢醌用量;但由于麦秸的“氮因子效应”又固定了尿素分解产物及其氧化产物,从而弥补了氢醌失效后可能造成氮素的继续损失。     

寄主大豆对根结瘤的控制

我国９个大豆（ＧｌｙｃｉｎｅｍａｘＬ．Ｍｅｒｒ．）品种感染根瘤菌ＵＳＤＡ１１０后,产生不同的结瘤数,低者在２０个以下．高者在６０个以上。赤豆、绿赤豆也可被感染结瘤,而豇豆、扁豆则不能。超结瘤大豆ｎｔｓ３８２作为接穗时能诱导我国大豆原结瘤数有４５个的开育１０号、原结瘤数有１２个的大黄分别发生高结瘤。ｎｔｓ３８２作为砧木时,则不能表现超结瘤．表明超结瘤因子能传给我国大豆,反之存在于我国大豆中的限制超结瘤的因子也能传给ｎｔｓ３８２。ｎｔｓ３８２于ＮＯ３－环境中仍表现超结瘤的特点也能导入开育１０号、大黄及赤豆根部,并使之在ＮＯ３－环境中结瘤。在ＮＯ３－环境中不能结瘤的开育１０号作为接穗,ｎｔｓ３８２作为砧木的嫁接植株,于子叶生长阶段接受ＮＯ３－时,仍能结瘤,于真对生长时接受ＮＯ３－时．则不能结瘤,表明限制结瘤因子于真叶细胞中被诱导形成。     

Primary Analysis of Components in Different Soybean Nodulating Mutants and their Effects on Nitrate Reductase Activity and Nodulation

The water extracts of leaves and roots from supernodulating soybean (Glycine max (L.) Merr. ) nts 382 and nonnodulating soybean Nod 49 have been chromatographed using filtering method through the column (25 cm × 2 cm) Sephadex G25 and 4 fractions, namly, nts 382 (Nod 49) F1, nts 382 (Nod 49) F2, nts 382 (Nod 49) F3, and nts 382 (Nod 49) F4 could be distinguished according to nitrate reductase (NR) activities inhibited by the eluate. The inhibition of NR activity by the noninoculated nts 382 F2 and the nts 382 F4 in vitro were much stronger than that by the inoculated nts 382 F2 and nts 382 F4. On the contrary, the obvious inhibition of NR activity in vitro by the noninoculated Nod 49 F2 and Nod 49 F4 were substantialy strengthed again by the innoculated Nod 49 F2 and Nod 49 F4. The facts indicated that the quantity of NR inhibitors in the leaf cells of soybean nts 382 reduced after the inoculation but was that in the inoculated Nod 49 leaf cells further more accumulated. Both nodulations assays, the nodulation of soybean "Bragg " injected with inoculated nts 382 Fl, nts 382 F2, nts 382 F3 and nts 382 F4 from leaves and roots and the nodulation of soybean nts 382 injected with inoculated Nod 49 F2, Nod 49 F3 and Nod 49 F4 from leaves only showed that nts 382 Fl and nts 382 F2 increased nodules of soybean "Bragg" by 1 to 3 times but nts 382 F3 and nts 382 F4 did not. Inhibition of soybeannts 382 nodulation by inoculated Nod 49 F2 Nod 49 F3 and Nod 49 F4 expressed that the Nod 49 F4 only inhibited the nodulation strongly by one time in the experiments with nts 382 plants with leaves, and by 15 times in the experiments with nts 382 plants without leaves at 10 d of inoculation and injection and this inhibition was nonreversible even after stopping injection from the 11th day to the 15th day after inoculation.     

Comparative Proteomic Analysis of Soybean Nodulation Using a Supernodulation Mutant,SS2-2

Legumes have the ability to form root nodules that fix atmospheric nitrogen through a symbiotic interaction with nitrogen-fixing bacteria. As a first step in dissecting the molecular process of nodulation, proteome reference maps of soybean roots and nodules were constructed. Time course analysis revealed that the transition from root to nodule was accompanied with downregulation of defense-response related proteins, including Mn-superoxide dismutase, peroxidase (Prx), PR10, and stress-induced protein, leading to the initiation of a symbiotic interaction between the two partners. Following nitrogenase biosynthesis, the host plant cooperated with the rhizobia to fix atmospheric nitrogen under microaerobic conditions via expression of leghemoglobins and antioxidant proteins. Comparative proteome analysis indicated lower expression of malate dehydrogenase (MDH), leghemoglobins and nitrogenase in the nodule development of the supernodulation mutant, SS2-2, as compared to the wild type, indicating that SS2-2 forms functionally immature nodules in higher numbers with the lower activity of nitrogen fixation.     

OsPT6基因过表达对低磷条件下菜用大豆结瘤及固氮的影响

采用砂培方法,以转OsPT6基因的菜用大豆(T3株系)与其非转基因(NT)受体品种为实验材料,研究了两者在低磷条件下的生长发育指标,植株有效磷、全磷、全氮、豆血红蛋白和籽粒蛋白质含量以及谷氨酰胺合成酶活性的差异,并对植株结瘤及固氮相关基因表达进行检测,为阐明转OsPT6基因菜用大豆在低磷条件下结瘤及固氮相关机理提供理论依据。结果显示:(1)转基因植株的株高、茎粗、花数和荚数、根瘤数均显著高于NT植株。(2)转基因植株根、茎、叶及根瘤中有效磷,全株总磷、总氮含量,根瘤中的豆血红蛋白含量、功能叶片中谷氨酰胺合成酶的活性和籽粒蛋白质含量均显著高于NT植株。(3)相关性分析表明,豆血红蛋白含量、谷氨酰胺合成酶活性、总磷、总氮含量4个指标间均呈显著正相关关系。(4)GmENOD40a、GmENOD40b、GmGS1β1、GmGS1β2基因在转基因植株中的表达量显著高于NT植株。研究表明,OsPT6基因过表达增强了菜用大豆在低磷条件下的结瘤及固氮能力,该研究结果为进一步研究其调控机理奠定了基础。     

一种高效研究大豆根瘤共生固氮的营养液栽培体系

为建立一种既可高效结瘤固氮, 又具有一定产量的大豆(Glycine max)营养液栽培系统, 设计并进行了2个试验。首先在不同供氮条件下, 研究了接种根瘤菌对大豆的结瘤状况、固氮能力、生物量及产量的影响。结果表明, 供氮过高或过低, 均影响大豆生长、产量形成及根瘤固氮; 并且植物生长所需的最适供氮水平远高于生物固氮所需的最适供氮水平。此外, 大豆生物固氮活性最高的时期在生殖期第一期(R1期)之前。由此推断, 大豆R1期前, 供应较低的氮, 有利于根瘤形成及固氮; 而从R1期起, 应提高供氮水平, 以促进植物生长及产量的形成。在此基础上开展第2个试验, 对供氮条件进行了优化处理(即R1期前低氮供应、R1期开始中氮供应)。结果表明, 与持续供应高氮相比, 优化供氮处理不仅可获得较多固氮酶活性较高的大根瘤, 还能保持较好的生长、获得更高的百粒重及维持80%左右的产量。研究结果不仅可为高效研究大豆根瘤共生固氮提供营养液配方, 还可为大豆高产高效栽培提供试验依据。     

Effects of Oxygen Concentration and Leghemoglobin on Organic Acid Degradation by Isolated Soybean Nodule Bacteriods

Bacteroids retaining high acetylene reduction activity (nitrogenase activity) were prepared anaerobically from soybean nodules. Addition of succinate (or of both leghemoglobin and succinate) to the acetylene reduction assay system greatly increased the activity of the isolated bacteroids.

When various organic acids were incubated with the bacteroids at 2% oxygen concentration, an optimum condition for bacteroid acetylene reduction, the organic acid degradation by bacteroids was very slow, and both lactate and acetate were accumulated in the incubation system, suggesting the operation of fermentative pathway in bacteroids under such low oxygen conditions.

With 20% oxygen, the added organic acids were degraded rapidly by bacteroids without addition of leghemoglobin to the incubation system.

With leghemoglobin in the incubation system, the organic acid degradation by bacteroids was accelerated extensively even at 2% oxygen, and the formation of lactate and acetate were negligible. No significant difference in the organic acid degradation rate was observed between the 2% and 20% oxygen concentrations when the leghemoglobin was present in the incubation system. Addition of acetylene to the assay system slightly inhibited the organic acid degradation.

This data suggests that bacteroids are unable to oxidize organic acid in low oxygen concentration and that the leghemoglobin allows the rapid organic acid dagradation by bacteroids even in such low oxygen concentrations.     

The Effect of CO2 Enrichment on the Growth of Nodulated and Non-Nodulated Isogenic Types of Soybean Raised Under Two Nitrogen Concentrations

To find the effects of CO₂ enrichment on plant development and photosynthetic capacity of nodulated (line A62-1) and non-nodulated (line A62-2) isogenic lines of soybean (Glycine max Merr.), we examined the interactions among two CO₂ treatments (36±3 Pa = AC and 70±5 Pa = EC), and two nitrogen concentrations [0 g(N) m⁻²(land area) = 0N; 30 g(N) m⁻²(land area) = 30N]. Nodules were found in both CO₂ treatments in 0N of A62-1 where the number and dry mass of nodules increased from AC to EC. While the allocation of dry mass to root and shoot and the amount of N in each organ did not differ between the growth CO₂ concentrations, there was larger N allocation to roots in 0N than in 30N for A62-2. The CO₂-dependence of net photosynthetic rate (P _N) for A62-1 was unaffected by both CO₂ and N treatments. In contrast, the CO₂-dependence of P _N was lower in 0N than in 30N for A62-2, but it was independent of CO₂ treatment. P _N per unit N content was unaffected by CO₂ concentrations. The leaf area of both soybean lines grown in 30N increased in EC. But in 0N, only the nodulated A62-1 showed an increase in leaf area in EC. Nitrogen use efficiency of plants, NUE [(total dry mass of the plant)/(amount of N accumulated in the plant)] in 30N was unaffected by CO₂ treatments. In 0N, NUE in EC was lower than in AC in A62-1, and was higher than that at AC in A62-2. Hence, the larger amount and/or rate of N fixation with the increase of the sink-size of symbiotic microorganisms supplied adequate N to the plant under EC. In EC, N deficiency caused the down-regulation of the soybean plant. This revised version was published online in June 2006 with corrections to the Cover Date.     

Nodule numbers are governed by interaction between CLE peptides and cytokinin signaling

CLE peptides are involved in the balance between cell division and differentiation throughout plant development, including nodulation. Previously, two CLE genes of Medicago truncatula, MtCLE12 and MtCLE13, had been identified whose expression correlated with nodule primordium formation and meristem establishment. Gain-of-function analysis indicated that both MtCLE12 and MtCLE13 interact with the SUPER NUMERIC NODULES (SUNN)-dependent auto-regulation of nodulation to control nodule numbers. Here we demonstrate that cytokinin, which is essential for nodule organ formation, regulates MtCLE13 expression. In addition, simultaneous knockdown of MtCLE12 and MtCLE13 resulted in an increase in nodule number, implying that both genes play a role in controlling nodule number. Additionally, a weak link may exist with the ethylene-dependent mechanism that locally controls nodule number.