利用低温水浴鉴定水稻苗期耐寒性

高效准确鉴定苗期耐寒性是水稻(Oryza sativa)耐寒研究的前提。基于流动水浴温度均一这一特性, 建立了一种恒温水浴鉴定水稻幼苗耐寒性方法。该方法中环境温度设定为20°C, 水浴温度设定为4°C。根据对2个水稻亚种不同材料的处理结果, 总结出几种常见品种的低温处理时间与存活率参考值, 并对操作过程中的一些注意事项进行了说明。     

一株水稻纹枯菌拮抗细菌的分离与鉴定

【目的】从土壤中分离并鉴定水稻纹枯菌拮抗细菌,测定其体外抑菌和温室防治效果。【方法】采用系列稀释法和平板对峙法筛选拮抗细菌,基于形态、生理特征及16S rDNA序列鉴定其分类地位,采用种子细菌化温室试验测定其防效。【结果】从蔬菜根际土壤中筛选出一株纹枯菌拮抗细菌,命名为kwkjT4。菌株具有明显的体外抑菌活性,对水稻纹枯病的温室防效与井冈霉素相当,初步鉴定为假紫色色杆菌(Chromobacterium pseudoviolaceum)。最适生长条件为pH 7.0,温度32°C,培养时间为36 h;抑菌活性物质产生的最适培养条件为pH 6.0,温度28°C,培养时间为48 h;表明两者并不一致。【结论】kwkjT4菌株在水稻纹枯病的生物防治中具有潜在的应用价值。这是C.pseudoviolaceum拮抗纹枯菌的首次报道。     

次氯酸钠离析法观测甘蓝叶片脉序的处理条件优化筛选

次氯酸钠（NaClO）离析法主要用于植物叶片表皮的观测,在研究过程中发现该法也可用于叶片脉序的观测。以甘蓝（Brassica oleracea var.capitata）为实验材料,采用二次正交设计方法对水煮时间、NaOH浓度、NaOH处理时间、NaClO浓度、NaClO处理温度和处理时间等各种处理条件进行优化筛选,以期得到适合于甘蓝叶片脉序观测的最佳处理条件组合。实验结果表明,新鲜甘蓝叶片水煮3分钟,10%的NaOH溶液60°C水浴处理2.5小时,3%的NaClO溶液40°C水浴离析2小时,叶片脉序的观测效果最佳。     

应用便携式数字化温控烫伤仪建立标准化大鼠皮肤烫伤模型

目的:研制一种新型便携式数字化温控烫伤仪,通过自制数字化烫伤仪对大鼠进行烫伤实验,建立标准化大鼠皮肤烫伤模型,从而验证数字化烫伤仪的有效及实用性。方法:选取18只SD大鼠分为对照组(3只,不烫伤);实验组(15只),再将实验组大鼠随机分配为A组(6只)、B组(6只)、C组(3只),选取大鼠背部作为烫伤实验部位:1、时间设定为20 s和30 s,烫伤仪温度分别设定为70℃、80℃、90℃、100℃、120℃,对A组和B组大鼠进行烫伤处理。2、温度设定为100℃,烫伤仪加热时间分别设定为10 s、15 s、25 s,对C组大鼠进行烫伤处理。烫伤后48 h切取实验组和对照组大鼠背部皮肤,进行大体及显微镜下组织病理学观察。结果:70℃20s烫伤点为I°烫伤;70℃30 s、80℃20 s、100℃10 s烫伤点为浅Ⅱ°烫伤;80℃30 s和100℃15 s烫伤点为深Ⅱ°烫伤;90℃20 s、90℃30 s、100℃20 s烫伤点为Ⅲ°烫伤;100℃25 s、100℃30 s、120℃20 s、120℃30 s烫伤点为IV°烫伤。结论:新型便携式数字化温控烫伤仪,可以可靠的建立标准化大鼠皮肤烫伤模型。     

一株产木聚糖酶嗜热菌的鉴定及酶学性质

从云南腾冲热泉水样中分离筛选得到一株产木聚糖酶的菌株。通过细菌形态观察、生理生化特性并结合16S rDNA序列分析,经鉴定,该菌为地芽孢杆菌(Geobacillus sp.),命名为Geobacillus sp.PZH1。对该菌株所产嗜热木聚糖酶及酶学特性进行了初步研究。SDS-PAGE和酶谱分析测得该酶分子量约为69 kD;酶的最适反应pH和最适反应温度分别为7.0和70°C,在pH 5.0-11.0和40°C-100°C范围内均有较高酶活;该酶在pH 5.0-12.0范围内和70°C以内具有较高的稳定性;40°C-100°C范围内,该木聚糖酶没有被检测到纤维素酶活性。     

来源于疣粒野生稻的越冬性新种质SH5的鉴定

低温冷害是影响水稻稳产的重要限制因子之一,鉴定和发掘利用优异的耐寒水稻种质资源是选育耐冷水稻品种以减少因低温对水稻生产不利影响的一条经济有效的策略。本研究通过让正季种植的水稻植株自然越冬翌年观察是否长出再生苗的方式鉴定了一份来源于疣粒野生稻的越冬性水稻新种质SH5;0℃低温处理结果表明,SH5的越冬性与其地上部植株的耐寒性无关;田间越冬性观察表明,SH5以地表及其以下的茎越冬;SH5越冬再生苗的扦插种植具有与正季种植的SH5相近的农艺性状。越冬性水稻新种质SH5的鉴定,为选育生产上应用的耐冷水稻品种提供宝贵的新种质资源。     

水稻孕穗期耐热种质资源的初步筛选

在人工气候室设定极端高温条件下,对来源于不同国家和地区的58份处于温度敏感期的水稻品种耐热性进行鉴定,以结实率下降的百分率为指标,通过方差分析,对水稻种质资源的耐热性进行评价。结果表明,种质间的耐热性存在显著差异,筛选出极端耐热品种2份（IRAT118和冷水白）。     

高温海藻床中微生物的分离鉴定及其耐热、耐盐特性研究

【目的】对印度洋卡利安达岛海洋热泉周边的高温海藻床中的微生物进行分离培养和16S rDNA种属鉴定,并研究其耐热和耐盐特性。【方法】采集海藻床中马尾藻、沉积物和海水样品,采用MGYTC培养基,分别于55°C和30°C对样品中的微生物进行培养和分离纯化;采用16S rDNA鉴定种属并构建进化树;研究培养温度和盐度对几种菌株生长的影响。【结果】共获得4个纲、9个属的12种菌株,与已知种属的相似度高于98%。芽孢杆菌纲嗜热放线菌科2个属的菌株和芽孢杆菌属菌株在55°C培养时获得,为嗜热菌株;其中高温放线糖莱斯菌(Laceyella sacchari)和热噬淀粉芽孢杆菌(Bacillus thermoamylovorans)可以在0 90的盐度范围生长。γ-变形菌纲希瓦氏属的Shewanella upenei、Shewanella algidipiscicola和鲍希瓦氏菌(Shewanella haliotis)可适应30°C 55°C的生长环境。【结论】研究获得的菌株,具有耐热、耐盐或对温度适应范围广等特性,有望成为生物技术领域的新资源;分离获得的Tepidibacter formicigenes,深海微小杆菌(Exiguobacterium profundum)和魔鬼弧菌(Vibrio diabolicus)曾被报道来源于深海热液区,这对于阐明海洋热泉与深海热液环境的内在联系提供了参考。     

一株兼性氧化亚氮还原菌的还原N2O能力

【目的】从水稻土中分离筛选出一株兼性氧化亚氮还原菌,并探索其在不同条件下还原N_2O的能力,为减少温室气体N_2O的排放提供重要依据。【方法】通过微生物富集培养分离技术从水稻土中分离得到纯菌;利用nosZ基因和16S rRNA的测序分析鉴定菌株;通过测定菌株在不同条件下N_2O的还原量,分析该菌株还原N_2O的能力及调控因子。【结果】经鉴定,该菌株含有nos Z基因,属于假单胞菌属,在温度30°C、厌氧条件下还原N_2O速率高达0.0219μmol/min以上,改变不同温度和氧气浓度后其能力相对减弱,但仍具备较强的还原N_2O作用。【结论】从水稻土中分离筛选得到的兼性氧化亚氮还原菌为假单胞菌,它在不同环境条件下都具备较强的还原N_2O能力,该菌株可能为减少土壤N_2O排放提供新途径,对保障生态环境安全具有重要的应用价值。     

一株海洋低温葡萄糖氧化酶菌株的筛选、鉴定及部分酶学性质

【目的】从海洋样品中分离筛选出产葡萄糖氧化酶菌株。【方法】采用双层平板筛选法进行初筛、复筛确定一株酶活较好的菌株,命名为GOD2(Glucose oxidase)。通过形态学、生理生化特征及16S rRNA基因序列分析研究其分类地位,并对其产生的葡萄糖氧化酶进行分离纯化和部分酶学性质的研究。【结果】细菌GOD2为产葡萄糖氧化酶菌株且遗传稳定,初步鉴定该菌株为假单胞杆菌(Pseudomonas migulae),其所产酶最适反应温度为20°C,热稳定性较差,40°C剩余相对酶活80%;超过40°C酶活力迅速下降。【结论】GOD2是一株极具研究价值的产低温葡萄糖氧化酶菌株。目前没有关于利用该菌生产葡萄糖氧化酶的报道。     

Cold Stress-Induced Acclimation in Rice is Mediated by Root-Specific Aquaporins

Cold acclimation process plays a vital role in the survival of chilling- and freezing-tolerant plants subjected to cold temperature stress. However, it remains elusive whether a cold acclimation process enhances root water uptake (a component of chilling tolerance) in chilling-sensitive crops such as rice. By analyzing the root hydraulic conductivity under cold stress for a prolonged time, we found that cold stress induced a gradual increase in root osmotic hydraulic conductivity [Lp(r(os))]. Compared with the control treatment (roots and shoots at 25°C), low root temperature (LRT) treatment (roots at 10°C; shoots at 25°C) dramatically reduced Lp(r(os)) within 1 h. However, Lp(r(os)) gradually increased during prolonged LRT treatment and it reached 10-fold higher values at day 5. Moreover, a coordinated up-regulation of root aquaporin gene expression, particularly OsPIP2;5, was observed during LRT treatment. Further, comparison of aquaporin gene expression under root-only chilling (LRT) and whole-plant chilling conditions, and in the roots of intact plants vs. shootless plants, suggests that a shoot to root signal is necessary for inducing the expression of aquaporin genes in the root. Collectively, these results demonstrate that a cold acclimation process for root water uptake functions in rice and is possibly regulated through aquaporins.     

New insights into the genetic basis of natural chilling and cold shock tolerance in rice by genome‐wide association analysis

In order to understand cold adaptability and explore additional genetic resources for the cold tolerance improvement of rice, we investigated the genetic variation of 529 rice accessions under natural chilling and cold shock stress conditions at the seedling stage using genome‐wide association studies; a total of 132 loci were identified. Among them, 12 loci were common for both chilling and cold shock tolerance, suggesting that rice has a distinct and overlapping genetic response and adaptation to the two stresses. Haplotype analysis of a known gene OsMYB2, which is involved in cold tolerance, revealed indica–japonica differentiation and latitude tendency for the haplotypes of this gene. By checking the subpopulation and geographical distribution of accessions with tolerance or sensitivity under these two stress conditions, we found that the chilling tolerance group, which mainly consisted of japonica accessions, has a wider latitudinal distribution than the chilling sensitivity group. We conclude that the genetic basis of natural chilling stress tolerance in rice is distinct from that of cold shock stress frequently used for low‐temperature treatment in the laboratory and the cold adaptability of rice is associated with the subpopulation and latitudinal distribution.     

东乡野生稻苗期耐冷性的遗传分析

以强耐冷材料龙稻5号（Oryza sativa ssp.japonica）和冷敏感材料协青早B（O.sativa ssp.indica）为对照品种,以萎蔫率和死苗率为鉴定指标,利用江西东乡普通野生稻（O.rufipogon）/协青早B//协青早B构建的回交重组自交系（backcross in-bred lines,BILs）群体（BC1F9）进行苗期耐冷性鉴定和遗传分析。结果表明,10°C冷处理7天后,228个BILs群体的平均萎蔫率为67.4%,平均死苗率为70.8%。将死苗率≤20%的株系,在更低温度（8°C昼/5°C夜）下处理5天,结果显示5243和5335为强耐冷材料,可用于构建东乡野生稻QTL（quantitative traitloci）近等基因系（near-isogenic lines,NILs）。实验结果显示,群体萎蔫率和死苗率均呈偏态的连续分布,暗示东乡野生稻苗期耐冷性表现为质量-数量性状遗传特征,由主基因-多基因控制。     

低温对杂交水稻及其亲本三系始穗期旗叶光合作用的影响

研究了低温对5套杂交水稻组合的影响。始穗期旗叶光合作用的最大速率和表观量子效率受低温抑制的程度表明:其耐冷顺序为粳稻型的“秀优57”>“釉稻型的青优早”>“汕优63”、“汕优64”与“威优64”。杂交水稻 F_1的耐冷水平与母本近似,与父本关系不大,保持系的耐冷性相似于不育系。杂交水稻幼苗经低温处理后的存活率亦表现出同样的规律。证明所试5套杂交水稻的生育后期与苗期对低温的反应是一致的。本文对该结果在杂交水稻抗冷性选育种中的意义进行了讨论。     

东北中部地区水稻不同生育时期低温处理下生理变化及耐冷性比较

摘要：研究水稻不同生育时期低温条件下各种生理指标的变化,选育耐低温水稻新品种,对于东北地区的水稻生产具有重要意义。以东北中部地区目前主要栽培的15个主要栽培水稻品种为实验材料,利用人工气候箱进行低温处理,分析苗期、孕穗期、抽穗期和灌浆期4个不同生育时期各个品种过氧化物酶（POD）、丙二醛（MDA）、脯氨酸和叶绿素4种生理指标含量变化与耐低温系数之间的关系。同时利用不同生理指标隶属函数的加权平均值（D值）对不同水稻品种的抗冷性进行分类划分。结果表明,每个生育时期的低温处理下,过氧化物酶（POD）、脯氨酸、叶绿素均呈下降趋势,丙二醛（MDA）则呈上升趋势,并且4种指标的变化幅度与水稻耐低温系数之间呈极显著相关性（P＜0.01）,各种指标的加权平均值（D值）与抗低温系数之间相关系数达到极显著,并且高于每一个单一指标。因此,D值可以更有效的对水稻进行抗低温评价和分级。     

Biochemical identification of the OsMKK6-OsMPK3 signalling pathway for chilling stress tolerance in rice

MAPK (mitogen-activated protein kinase) pathways have been implicated in stress signalling in plants. In the present study, we performed yeast two-hybrid screening to identify partner MAPKs for OsMKK (Oryza sativa MAPK kinase) 6, a rice MAPK kinase, and revealed specific interactions of OsMKK6 with OsMPK3 and OsMPK6. OsMPK3 and OsMPK6 each co-immunoprecipitated OsMKK6, and both were directly phosphorylated by OsMKK6 in vitro. An MBP (myelin basic protein) kinase assay of the immunoprecipitation complex indicated that OsMPK3 and OsMPK6 were activated in response to a moderately low temperature (12°C), but not a severely low temperature (4°C) in rice seedlings. A constitutively active form of OsMKK6, OsMKK6DD, showed elevated phosphorylation activity against OsMPK3 and OsMPK6 in vitro. OsMPK3, but not OsMPK6, was constitutively activated in transgenic plants overexpressing OsMKK6DD, indicating that OsMPK3 is an in vivo target of OsMKK6. Enhanced chilling tolerance was observed in the transgenic plants overexpressing OsMKK6DD. Taken together, our data suggest that OsMKK6 and OsMPK3 constitute a moderately low-temperature signalling pathway and regulate cold stress tolerance in rice.     

Effect of Chilling on Photosynthesis in Flag Leaves at Primary Heading Stage of F1 Hybrid Rice Cultivars and Their Parental Lines (Three Lines)

With the purpose of selecting and breeding cold stress tolerance of hybrid rice cultivars, the effect of chilling on five hybrid rice combinations was investigated. The results indicated that the cold tolerance of different hybrid rice varieties were different. The order of the cold tolerance as the degrees of inhibition of maximum photosynthetic rate and of apparent quantum yield of flag leaves at primary heading stage by chilling was as follows: The cold tolerance of Japanica type Xiuyou-57 was the highest then lndica type Qing-Youzao followed by Shangyou-63, Shangyou-64 and Weiyou-64. The cold tolerance of Fx hybrid rice was similar to that of the maternal lines and not similar to the paternal lines. The cold tolerance of maintainer lines was similar to that of the male sterile lines. The tests on the survival rate of hybrid rice seedlings after chilling treatment also showed a similar re gular patterns. These indicated that the responses by the chilling temperature in these five hybrid combinations were exactly the same in the Seedling stage as well as in the late growing stage. The results of these experiments on the impact of selecting and breeding cold tolerance in hybrid rice were discussed.     

Physiological and morphological characteristics of chickpea accessions under low temperature stress

Economically important crop chickpea (Cicer arietinum L.) is sensitive to chilling stress, and breeding for chilling tolerance is the economic option even in countries with a high risk for drought and heat stresses. In this study, we have analyzed chilling-induced responses of ten chickpea accessions under field and growth-chamber conditions in order to screen, using phenotypic and physiological methods, for chilling tolerance. The field data analysis revealed that there were significant differences between accessions in their cold tolerance. The percent survival and cold-tolerance scores were the most important indices describing genotype tolerance to low temperature under field conditions; they can be used to assess chickpea cold tolerance. During environmentally controlled testing, the effects of low temperature regimes (−10°C for 15 and 30 min) were studied and cold tolerance was measured by electrolyte leakage from damaged leaves. The analysis of field data and cold treatments showed that two accessions, Sel 95Th1716 and Sel 96Th11439, grouped in one cluster, are good cold-tolerant genotypes (showing low scores for cold tolerance and electrolyte leakage). In comparison with ILC 8262, released as cold-tolerant accession, these genotypes showed more tolerance. Flip 00-6C, ILC 533, and Jam were less tolerant to cold stress. Thus, we have shown that as well as field studies, short-term cold treatment and electrolyte leakage assay can be used to evaluate low temperature tolerance of chickpea profitably in a short time.     

黄瓜核心种质低温耐受性的田间评价

在自然低温条件下,利用幼苗存活率、冷害指数、叶片数、株高及叶绿素含量5个指标对303份黄瓜核心种质进行了低温耐受性评价,结果表明:5个指标基本符合正态分布的特点,幼苗存活率和冷害指数均有明显向低值和高值区域偏离的趋势。相关分析结果显示冷害指数与幼苗存活率、叶片数、株高及叶绿素含量4个指标呈极显著负相关。通过隶属度函数综合评价群体的低温耐受性,共筛选获得28份耐低温种质,为黄瓜耐低温的品种选育及进一步深入挖掘低温耐受性相关基因奠定了基础。