转OsMAPK4基因烟草的抗旱性研究与遗传分析

以低温处理的水稻辽盐241植株叶片总RNA为模板, 用OsMAPK4基因特异引物通过RT-PCR扩增出OsMAPK4基因, 构建了由E12启动子调控的OsMAPK4基因植物表达载体pBME12。通过农杆菌介导法将OsMAPK4基因导入烟草, 筛选获得25株转基因植株。抗旱性研究结果表明, OsMAPK4基因的超量表达提高了T1代转基因植株的抗旱性。卡那霉素抗性在T1代转基因植株中的分离情况表明, 大多数转基因株系符合单基因遗传规律。     

不同年代大豆品种根系活力的变化及其与植株生物量的关系

为探索不同年代大豆品种根系活力的变化及其与植株各器官生物量和根系伤流液重量的关系。以吉林省1923~2009年间育成的27个大豆品种为材料进行田间试验,在四叶期(V4),盛花期(R2),结荚盛期(R4)和鼓粒盛期(R6)测定根系活力、伤流液重量和植株各器官生物量。结果表明,随生育期的推进,植株器官生物量不断增加,而根系活力和伤流液重量则呈单峰曲线变化,在R4期达到最大。在R4期,植株根系活力、根系伤流液重量和器官生物量与育成年代均呈显著或极显著正相关,但在V4、R2和R6期均未达到显著水平。在R4期,根系活力与根系伤流液重量、植物器官茎、叶和根生物量呈显著或极显著正相关;各品种间地上部器官生物量与根系活力的比值差异不显著。大豆的遗传改良使地上部器官与根系活力协同演进,在根系活力增强的同时植株器官生物量和根系伤流液重量也增加,且在R4期,根系活力与植物器官生物量和根系伤流液重量相关最为密切;可以把R4期的根系活力作为高产育种的指标之一。     

杨树MYB转录因子家族基因应答盐胁迫表达特性分析

MYB转录因子家族是植物中数量最多的转录因子家族之一,在植物次生代谢调节、信号转导和抗逆等生物过程起重要作用。根据MYB转录因子结构域组成差异可分4个亚家族：即1R-MYB（MYB-relaed）、R2R3-MYB、3R-MYB和4R-MYB。其中,R2R3-MYB亚家族数量最多,可进一步分为22个亚组;利用生物信息学分析杨树MYB转录因子蛋白序列的保守结构域、系统发生、基因组定位、氨基酸组成和理化性质等;参照拟南芥MYB转录因子功能,预测杨树MYB转录因子功能;基于84K杨转录组测序和RT-qPCR分析,从301个杨树MYB转录因子基因中筛选出69个应答盐胁迫基因（P≤0.05）。其中,上调表达基因32个,下调表达基因37个。该研究可为进一步研究杨树MYB家族基因功能提供参考依据。     

Multiple shoots induction in wild cotton (<Emphasis Type="Italic">Gossypium bickii</Emphasis>) through organogenesis and the analysis of genetic homogeneity of the regenerated plants

Fertile plants were regenerated via organogenesis from cultures of Gossypium bickii Prokhanov cotyledonary nodes devoid of cotyledons. Cotyledonary nodes from 7-day-old seedlings yielded the maximum number of shoots (9.12 shoots per explant) using MSB medium [MS medium (Murashige & Skoog 1962) and B5 (Gamborg et al. 1968) vitamins] supplemented with the combination of 4 mg/L BAP (N6-benzyladinine) and 0.1 mg/L TDZ (thidiazouron), and the subculture was made on a medium containing 2 mg/L BAP. Elongation of multiple shoots was obtained on MSB medium containing 0.05 mg/L GA₃ (gibberellic acid). For rooting, a modified half-strength MS medium was utilized. Some regenerants were transferred to a greenhouse, and most of them were morphologically normal and fertile. Histological studies revealed that proliferating buds originated directly from the superficial layers of the explants. Flow cytometric analyses and chromosome counting suggested that the regenerants and their offsprings were diploid. DNA content of regenerants and their offsprings was homogeneous and similar to that of seed-derived plants with unchanged chromosome number (2n = 26). Random amplified polymorphic DNA (RAPD) analysis confirmed the genetic homogeneity of the regenerants and their offsprings with the diploid parent.     

中华蜜蜂热休克蛋白70的克隆、表达及中蜂囊状幼虫病毒感染后表达差异分析

[目的]本研究旨在明确中华蜜蜂Apis cerana cerana热休克蛋白70 (Heat shock protein 70,HSP70)与中蜂囊状幼虫病毒(Chinese sacbrood virus,CSBV)的关系.[方法]首先根据NCBI上已公布的中华蜜蜂HSP70的全基因(NO.MH122655.1)序列,设计特异性引物,提取3日龄中华蜜蜂幼虫总RNA,通过RT-PCR方法获得HSP70基因,并通过生物信息学软件进行分析;其次将其克隆到原核表达载体pET-32a中,转化入大肠杆菌BL21(DE3)进行蛋白表达,并制备重组蛋白,将纯化的重组蛋白免疫小鼠制备多克隆抗体;最后利用制备多克隆抗体作为一抗,通过Western-blot方法检测CSBV感染中华蜜蜂前后HSP70在幼虫体内的变化.[结果]成功获取中华蜜蜂HSP70蛋白的全基因,其核苷酸序列全长为1833 bp,生物信息学分析后,发现其编码蛋白存在7个抗原表位.利用原核表达系统,成功获得大小约87 ku的重组蛋白HSP70,免疫小鼠后获得多克隆抗体,经Western-blot分析能与标签蛋白发生特异性反应;对感染CSBV前后蜜蜂体内HSP70蛋白检测发现感染后HSP70表达水平显著提高.[结论]通过原核表达系统,成功表达中华蜜蜂HSP70基因和HSP70多克隆抗体,并证实CSBV感染后影响中华蜜蜂体内HSP70蛋白表达量的改变,为深入研究HSP70功能提供了帮助.     

Changes in nitric oxide, cGMP, and nitrotyrosine concentrations over skin along the meridians in obese subjects

The purposes of these studies were to quantify the concentrations of total nitrate and nitrite (NO(x)(-)) cyclic guanosine monophosphate (cGMP), and nitrotyrosine over skin surface in normal weight healthy volunteers (n = 64) compared to overweight/obese subjects (n = 54). A semi-circular plastic tube was taped to the skin along acupuncture points (acupoints), meridian line without acupoint (MWOP), and nonmeridian control and filled with a 2-Phenyl-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl solution for 20 min. The concentrations of NO(x)(-), cGMP, and nitrotyrosine in the samples were quantified in a blinded fashion using chemiluminescence and enzyme-linked immunosorbent assay, respectively. In normal weight healthy volunteers, NO(x)(-) and cGMP concentrations were consistently increased over the pericardium meridian (PC) 4-7 compared with nonmeridian areas. NO(x)(-) concentration is enhanced over the bladder meridian (BL) 56-57, but cGMP level is similar between the regions. In overweight/obese subjects, NO(x)(-) contents were increased or tended to be elevated over PC and BL regions. cGMP is paradoxically decreased over PC acupoints and nonmeridian control on the forearm but the decreases were blunted along BL regions on the leg. Nitrotyrosine concentrations are markedly elevated (five- to sixfold) over both PC and BL in all areas of overweight/obese subjects. This is the first evidence showing that nitrotyrosine level is tremendously elevated over skin accompanied by paradoxical changes in nitric oxide (NO)-cGMP concentrations over PC skin region in overweight/obese subject. The results suggest that NO-related oxidant inflammation is systemically enhanced while cGMP generation is impaired over PC skin region but not over BL region in obesity.     

Pretreatment of microcrystalline cellulose in organic electrolyte solutions for enzymatic hydrolysis

Background

Biomass use for the production of bioethanol or platform chemicals requires efficient breakdown of biomass to fermentable monosaccharides. Lignocellulosic feedstocks often require physicochemical pretreatment before enzymatic hydrolysis can begin. The optimal pretreatment can be different for different feedstocks, and should not lead to biomass destruction or formation of toxic products.

Methods

We examined the influence of six mild sulfuric acid or water pretreatments at different temperatures on the enzymatic degradability of sugar-beet pulp (SBP).

Results

We found that optimal pretreatment at 140°C of 15 minutes in water was able to solubilize 60% w/w of the total carbohydrates present, mainly pectins. More severe treatments led to the destruction of the solubilized sugars, and the subsequent production of the sugar-degradation products furfural, hydroxymethylfurfural, acetic acid and formic acid. The pretreated samples were successfully degraded enzymatically with an experimental cellulase preparation.

Conclusions

In this study, we found that pretreatment of SBP greatly facilitated the subsequent enzymatic degradation within economically feasible time ranges and enzyme levels. In addition, pretreatment of SBP can be useful to fractionate functional ingredients such as arabinans and pectins from cellulose. We found that the optimal combined severity factor to enhance the enzymatic degradation of SBP was between log R'₀ = -2.0 and log R'₀ = -1.5. The optimal pretreatment and enzyme treatment solubilized up to 80% of all sugars present in the SBP, including ≥90% of the cellulose.     

γ干扰素释放试验与结核菌素皮肤试验筛查预测活动性结核病的比较

为评估γ干扰素释放试验（T-SPOT ．TB）和结核菌素皮肤试验（TST ）在我国活动性肺结核病患者的家庭接触者中预测活动性结核病的能力,选取157例家庭接触者行T-SPOT ．TB和TST筛查,并前瞻性随访6年。将121例参与随访的家庭接触者纳入分析,T-SPOT ．TB阳性率为49．6％,与年龄﹝ OR＝1．03（95％CI 1．00～1．05）,P＝0．032﹞和接触菌阳患者﹝ OR＝2．13（95％ CI为1．01～4．51）,P＝0．048﹞呈正相关。TST≥5 mm和TST≥10 mm的接触者分别占57．5％和37．2％,其中TST≥10 mm与接触菌阳患者呈正相关﹝ OR＝2．30（95％ CI为1．05～5．06）,P＝0．038﹞。共随访626人年,7例发展为活动性结核病,发病率为11．0／1000人年。T-SPOT ．TB的阳性预测值（PPV）为8．3％,阴性预测值（NPV）为96．7％。TST≥5 mm和TST≥10 mm的PPV分别为7．7％和9．5％,NPV均为95．8％。总之,活动性肺结核病患者的家庭接触者发展为活动性结核病的风险高于一般人群,而T-SPOT ．TB与TST对活动性结核病的预测能力相当。     

土壤pH值对烤烟叶片生理生化特性的影响

烟草生长初期,超氧化物歧化酶(SOD)活性及丙二醛(MDA)含量与pH增长呈正相关,而过氧化氢酶(CAT)和过氧化物酶(POD)活性则随着pH的增加而下降,pH值6.5和7.5时叶绿素含量和净光合速率(Pn)较高,旺长期达到高峰;生长后期,各处理的MDA含量和POD活性最高,SOD、CAT活性最低.各pH值处理的叶片中,叶绿素含量、Pn下降,比叶重达到高峰,但pH 8.5下的烟叶中叶绿素含量高,Pn大,比叶重较小.pH 6.5和7.5下的烟叶中蛋白质和可溶性糖总体含量高于其它pH的.烟碱含量在pH 5.5时最高,pH 8.5时最低.     

Purification and identification of a 42-kilodalton abscisic acid-specific-binding protein from epidermis of broad bean leaves

Purification of abscisic acid (ABA)-binding proteins is considered to constitute a major step toward isolating ABA receptors. We report here that an ABA-binding protein was for the first time, to our knowledge, purified from the epidermis of broad bean (Vicia faba) leaves via affinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing electrophoresis, and isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis two-dimensional electrophoresis of the purified ABA-binding protein all identified a single protein band with a molecular mass of 42 kD and an isoelectric point 4.86. The Scatchard plot for the purified protein showed a linear function with a maximum binding activity of 0.87 mol mol(-1) protein and an equilibrium dissociation constant of 21 nM, indicating that the purified protein may be a monomeric one, possessing one binding site. The ABA-binding protein was enriched more than 300-fold with a yield of 14%. (-)ABA and trans-ABA were substantially incapable of displacing (3)H-(+/-)ABA bound to the ABA-binding protein, and (+/-)ABA was less effective than (+)ABA in the competition. These findings allow establishment of the stereospecificity of the 42-kD protein and suggest its ABA receptor nature. Pretreatment of the guard cell protoplasts of broad bean leaves with the monoclonal antibody raised against the 42-kD protein significantly decreased the ABA specific-induced phospholipase D activity in a dose-dependent manner. This physiological significance provides more clear evidence for the potential ABA-receptor nature of the 42-kD protein.