Secretome analysis of thermophilic mould Myceliophthora thermophila cultivated on rice straw and hydrolysis of lignocellulosic biomass for bioethanol production

Abstract

Myceliophthora thermophila encodes for large number of carbohydrate-active enzymes (CAZymes) involved in lignocellulosic biomass degradation. The mould was grown on rice straw in solid state fermentation at pH 5.0 and 45?°C that produced high levels of cellulolytic and xylanolytic enzymes i.e. 2218.12, 515.23, 478.23, 13.34?U/g DMR for xylanase, CMCase, FPase and β-glucosidase, respectively. The secretome analysis of M. thermophila BJAMDU5 by mass spectroscopy, described 124 different proteins with majority of CAZymes consisting of glycosyl hydrolases (GH), lytic polysaccharide mono-oxygenases (LPMO), carbohydrate esterases (CE) and polysaccharide lyases (PL). Furthermore, the enzyme cocktail of the mould was evaluated for hydrolysis of steam treated rice straw that produced 184.59?mg/g substrate reducing sugars after 24?h, which was used for production of bioethanol by using fast fermenting yeast Saccharomyces cerevisiae resulting in high production of bioethanol.     

A complete sequence of the pGA1611 binary vector

We report the nucleotide sequence of the binary vector pGA1611, which is used for the transformation of foreign DNA into rice. This vector is 13,476 bp long. The 5577- bp T- DNA region consists of a 1987- bp ubiquitine promoter region, 45 bp for the multiple cloning site, a 253- bpnos terminator region, and the 2045- bpCaMV35S- hph- T7 chimaeric gene. The vector backbone (7004 bp) carriesoriT,traJ,trfA,tetA,tetR, andoriV. An 892- bp RB region and the 489- bp LB region are also present The T- DNA possesses 15 unique sites, six of which are at the multiple cloning site. This information will be valuable for cloning foreign DNA and modifying the vector.     

The first intron of rice EPSP synthase enhances expression of foreign gene

The first intron (EPI) of rice 5-enolpyruvylshikimate 3-phosphate synthase gene was isolated by PCR from one clone with genomic EPSP synthase gene. Sequence analysis showed that the first intron is 704 bp in length with 36.2% G+C content. To investigate its effect on expression of foreign gene, we inserted the first intron between CaMV35S promoter and β-glucuronidase (GUS) gene. The transient expression results showed that GUS could be expressed effectively with EPI. The GUS activity in transgenic tobacco shows that the EPI can greatly enhance the expression level of β-glucuronidase (P < 0.01) compared with transgenic tobacco without the first intron, and 3-to 6-fold increase in GUS activity in some transgenic tobaccos. Northern blot indicated the first intron was spliced from GUS pre-mRNA, and the steady-state mRNA levels of GUS with EPI in transgenic tobaccos were higher than that in transgenic tobacco without EPI, which suggested that the first intron of EPSP was a non-translated intron.     

水稻种子萌发期间谷氨酰胺合成酶和NADH-谷氨酸合酶的变化

测定了水稻种子不同萌发时期胚乳、胚芽鞘和幼根的谷氨酰胺合成酶（GS）和依赖于NADH的谷氨酸合酶（NADH－GOGAT）活性变化。胚乳和胚芽鞘的GS活性在萌发过程中升高,幼根的GS活性则有所降低。NADH－GOGAT的活性变化趋势与GS相同。Native-PAGE活性染色表明,在萌发阶段的水稻种子胚乳和幼根里,始终只观察到一种GS活性带。但是,在水稻种子萌发3d后,在胚芽鞘中除继续检测到GS1的活性外,还可以观察到GS2的活性。蛋白质印迹显示,水稻种子胚乳中的GS（GSe)和GS1和GSra一样是一种胞质型GS。实验结果提示,这些不同组织中的GS与NADH－GOGAT构成的循环途径也许是水稻种子萌发时氨同化的主要途径。     

Somaclonal variation and chilling tolerance improvement in rice: Changes in fatty acid composition

The relationship between chilling tolerance of six rice cultivars – Facagro 57, Facagro 76, Fujisaka 5, Kirundo 3, Kirundo 9 and IR64 -and the fatty acid composition in total lipids, phospholipids, galactolipids and neutral lipids from leaves was studied. Higher double bond index and proportions of linolenic acid in the phospholipid and galactolipid classes were related to cultivar chilling tolerance, but this was not so for the total lipids nor the neutral lipid class. The somaclonal families derived from Facagro 76, Kirundo 3 and Kirundo 9 that showed enhanced chilling tolerance as compared to their original parental cultivar were analyzed for fatty acid composition in phospholipids and galactolipids from leaves. Altered proportions in fatty acid composition in phospholipids, galactolipids or both were found in the somaclonal families derived from Facagro 76 and Kirundo 9, but not from Kirundo 3. These changes most usually resulted in higher double bond index and higher proportions in linoleic and linolenic acids which were related either to lower ratio of C16 to C18 fatty acids or to higher unsaturation in the C18 fatty acid fraction. Different mechanisms thus seem to be implicated in the altered fatty acid composition of somaclones, which may be related to the chilling tolerance improvement of some somaclonal families.     

2000年间野生稻和栽培稻(O.sativa.L)分布区逆向分离的过程及动力Ⅰ.古今野生稻和栽培稻(O.sativa.L)的分布与人口分布的关系

２０００ａ间,中国境内野生稻分布区域由２４９５５７５ｋｍ２缩减至１３７１０９４ｋｍ２,减少了４５．０６％;分布纬度北界由３８°３′Ｎ南移至２８°１４′Ｎ,南移９°４９′、１１４０ｋｍ;中国境内栽培稻分布区域由４０８１８６０ｋｍ２增加至９６０００００ｋｍ２,增加了１３５．００％;分布北界由３８°Ｎ北移至５３°２９′Ｎ,北移１５°２９′、１７００ｋｍ。人口分布重心主要在黄河流域和长江流域,对该区域野生稻的生存产生不利影响。而促进栽培稻的发展,必须建立野生稻自然保护区。     

秋稻品种‘Dular’广亲和基因效应的RFLP分析

利用RFLP标记对秋稻品种Dular的三交（南京11／／Dular/2533）的F2群体与灿粳品种的测交F1进行了单株带鉴定,并对广亲和性的表达进行分析。结果表明：与第6染色体上标记RG213连锁的广亲和基因S5^n效应较大;不同位点广亲和基因具有明显的累加效应;位点内的互作导致了粳型配子的部分败育,同时还存在位点间的互作。     

氢醌和双氰胺对种稻土壤N2O和CH4排放的影响

通过盆栽试验,研究了脲酶抑制剂氢醌（ＨＱ）、硝化抑制剂双氰胺（ＤＣＤ）及二者的组合（ＨＱ＋ＤＣＤ）对种稻土壤Ｎ２Ｏ和ＣＨ４排放的影响．结果表明,在未施麦秸粉时,所有施抑制剂的处理均较单施尿素的能显著减少水稻生长期供试土壤Ｎ２Ｏ和ＣＨ４的排放．特别是ＨＱ＋ＤＣＤ处理,其Ｎ２Ｏ和ＣＨ４排放总量分别约为对照的１／３和１／２．而在施麦秸粉后,该处理的Ｎ２Ｏ排放总量为对照的１／２,但ＣＨ４排放总量却较少差别．不论是Ｎ２Ｏ还是ＣＨ４的排放总量,施麦秸粉的都比未施的高出１倍和更多．因此,单从土壤源温室气体排放的角度看,将未腐熟的有机物料与尿素共施,并不是一种适宜的施肥制度．供试土壤的Ｎ２Ｏ排放通量,与水稻植株的ＮＯ－３Ｎ含量和土表水层中的矿质Ｎ量分别呈显著的指数正相关和线性正相关;ＣＨ４的排放通量则与水稻植株的生长量和土表水层中的矿质Ｎ量呈显著的线性负相关．在Ｎ２Ｏ与ＣＨ４的排放间,未施麦秸粉时存在着定量的相互消长关系;施麦秸粉后,虽同样存在所述关系,但难以定量化．     

取食转Bt基因水稻对稻纵卷叶螟幼虫中肠的组织病理学效应

利用透射电镜观察了稻纵卷叶螟 Cnaphalocrocis medinalis（Guenée）幼虫取食转Bt基因水稻后中肠的组织病理变化。结果表明：稻纵卷叶螟幼虫取食转cry1Ab基因水稻后,中肠上皮细胞的线粒体先发生形态变化,随连续取食时间的延长线粒体出现凝聚、内嵴稀疏、空泡化等,在后期还呈凝聚态随突起脱落或沿杯腔边沿单一排列。内质网的变化也很明显,病变过程中伴随着粗糙内质网的肿胀、核糖体脱落,粗糙内质网增多等现象。细胞核的变化较小,在处理后期出现细胞核拉长、核仁聚集等变化。组织病变程度不一,有的细胞在病变早期就出现了空泡化。