黑曲霉柠檬酸工业菌株原生质体制备与转化

黑曲霉是柠檬酸工业化生产的主要发酵菌株。尽管现代遗传操作技术在黑曲霉的实验室菌株、蛋白质生产菌株等的应用中获得了成功,但是柠檬酸工业菌株遗传转化异常困难,成为柠檬酸工业持续升级的重要限制因素。以柠檬酸工业生产实际应用的黑曲霉菌株为研究对象,对其原生质体的制备与再生以及PEG介导的转化等条件进行了细致优化。结果表明,柠檬酸高产工业菌株原生质体的制备需选取丰富培养基中培养48 h的年轻菌丝体,在1.5%裂解酶-0.5%蜗牛酶-0.2%溶菌酶的复合酶解体系下裂解2.5 h,原生质体的制备浓度可达106个/m L以上,原生质体再生效率可达90%以上。原生质体的浓度是原生质体-PEG介导转化方法的关键,当原生质体浓度达到106个/m L以上时,高产柠檬酸菌株的转化效率大幅提高。成功建立了由原生质体-PEG所介导的高产柠檬酸黑曲霉菌株的遗传转化体系。     

Time-Course Association Mapping of the Grain-Filling Rate in Rice (Oryza sativa L.)

Detecting quantity trait locus (QTLs) and elite alleles that are associated with grain-filling rate (GFR) in rice is essential for promoting the utilization of hybrid japonica rice and improving rice yield. Ninety-five varieties including 58 landraces and 37 elite varieties from the core germplasm collection were genotyped with 263 simple sequence repeat (SSR) markers. The GFR of the 95 varieties was evaluated at five stages, 7, 14, 21, 28 and 35 days after flowering (DAF) both in 2011 and 2012. We found abundant phenotypic and genetic diversity in the studied population. A population structure analysis identified seven subpopulations. A linkage disequilibrium (LD) analysis indicated that the levels of LD ranged from 60.3 cM to 84.8 cM and artificial selection had enhanced the LD. A time-course association analysis detected 31 marker-GFR associations involving 24 SSR markers located on chromosomes 1, 2, 3, 4, 5, 6, 8, 9, 11 and 12 of rice at five stages. The elite alleles for high GFR at each stage were detected. Fifteen excellent parental combinations were predicted, and the best parental combination ‘Nannongjing62401×Laolaihong’ could theoretically increase 4.086 mg grain^-1 d^-1 at the five stages. Our results demonstrate that the time-course association mapping for GFR in rice could detect elite alleles at different filling stages and that these elite alleles could be used to improve the GFR via pyramiding breeding.     

Land Use/Cover Change in the Middle Reaches of the Heihe River Basin over 2000-2011 and Its Implications for Sustainable Water Resource Management

The Heihe River Basin (HRB) is a typical arid inland river basin in northwestern China. From the 1960s to the 1990s, the downstream flow in the HRB declined as a result of large, artificial changes in the distribution of water and land and a lack of effective water resource management. Consequently, the ecosystems of the lower reaches of the basin substantially deteriorated. To restore these degraded ecosystems, the Ecological Water Diversion Project (EWDP) was initiated by the Chinese government in 2000. The project led to agricultural and ecological changes in the middle reaches of the basin. In this study, we present three datasets of land use/cover in the middle reaches of the HRB derived from Landsat TM/ETM+ images in 2000, 2007 and 2011. We used these data to investigate changes in land use/cover between 2000 and 2011 and the implications for sustainable water resource management. The results show that the most significant land use/cover change in the middle reaches of the HRB was the continuous expansion of farmland for economic interests. From 2000 to 2011, the farmland area increased by 12.01%. The farmland expansion increased the water resource stress; thus, groundwater was over-extracted and the ecosystem was degraded in particular areas. Both consequences are negative and potentially threaten the sustainability of the middle reaches of the HRB and the entire river basin. Local governments should therefore improve the management of water resources, particularly groundwater management, and should strictly control farmland reclamation. Then, water resources could be ecologically and socioeconomically sustained, and the balance between upstream and downstream water demands could be ensured. The results of this study can also serve as a reference for the sustainable management of water resources in other arid inland river basins.     

Blue light-dependent interactions of CRY1 with GID1 and DELLA proteins regulate gibberellin signaling and photomorphogenesis in Arabidopsis

Cryptochromes are blue light photoreceptors that mediate various light responses in plants and mammals. In Arabidopsis (Arabidopsis thaliana), cryptochrome 1 (CRY1) mediates blue light-induced photomorphogenesis, which is characterized by reduced hypocotyl elongation and enhanced anthocyanin production, whereas gibberellin (GA) signaling mediated by the GA receptor GA-INSENSITIVE DWARF1 (GID1) and DELLA proteins promotes hypocotyl elongation and inhibits anthocyanin accumulation. Whether CRY1 control of photomorphogenesis involves regulation of GA signaling is largely unknown. Here, we show that CRY1 signaling involves the inhibition of GA signaling through repression of GA-induced degradation of DELLA proteins. CRY1 physically interacts with DELLA proteins in a blue light-dependent manner, leading to their dissociation from SLEEPY1 (SLY1) and the inhibition of their ubiquitination. Moreover, CRY1 interacts directly with GID1 in a blue light-dependent but GA-independent manner, leading to the inhibition of the interaction between GID1 with DELLA proteins. These findings suggest that CRY1 controls photomorphogenesis through inhibition of GA-induced degradation of DELLA proteins and GA signaling, which is mediated by CRY1 inhibition of the interactions of DELLA proteins with GID1 and SCF^SLY1, respectively.

Blue light-dependent interactions of CRY1 with GID1 and DELLA proteins inhibit gibberellin (GA)-induced degradation of DELLA proteins to regulate GA signaling and photomorphogenesis.     

Sequence and expression analysis of the gene encoding inducible cAMP early repressor in tilapia

Suppression subtractive hybridization library was generated by comparison of cDNA populations isolated from peripheral leukocytes of pre- and post-immunized tilapia. One cDNA sequence encoding complete inducible cAMP early repressor was obtained from the library. The sequence was characterized by the presence of the basic structure of ICER IIγ. Expression of ICER was in the tissues of four types of tilapia was decreased after infection with Streptococcus. After immunization, expression of ICER was initially decreased and then increased after 7 days. In addition, the order for the overall expression of ICER gene after infection and the increases of ICER expression later after immunization in these four types of tilapia was positively correlated to the disease resistance and productivity of these four species of tilapia. Our results provided molecular mechanisms for the different disease resistance capability in different species of tilapia. In addition, our results also provided reference molecular marker for breeding disease resistant tilapia, cAMP responsive element modulator.     

RNA Interference-Based Transgenic Maize Resistant to Maize Dwarf Mosaic Virus

Maize dwarf mosaic virus (MDMV) is a widespread pathogenic virus that causes serious loss of yield in maize (Zea mays). RNA interference (RNAi) triggered by hairpin RNA (hpRNA) transcribed from a transgenic inverted-repeat sequence is an effective way to defend against viruses in plants. In this study, an hpRNA expression vector containing a sense arm and an antisense arm of 150 bp separated by an intron of the maize actin gene was constructed to target the P1 protein (protease) gene of MDMV and used to transform Agrobacterium tumefaciens strain EHA105. The transformed Agrobacterium strain was used to transform maize embryonic calli isolated from immature embryos by an improved culture technique. In all, 46 plants were regenerated after stringent hygromycin B selection, and 18 of them were certified to be positive by PCR amplification. Of these positive plants, 13 were grown to produce offspring, and nine were identified by Southern blotting to have the transgene integrated with one or two copies. The resistance of three T₂ lines was evaluated in a field trial of dual MDMV inoculation in two environments and was found to be improved compared with the non-transformed control. The disease indexes of the transgenic plant lines h2, 13, and h1 were not significantly different from the highly resistant control line H9-21. The viral titers of the inoculated plants were detected by double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), and the result was in accord with the resistance evaluated in the field trial. The addition of uniconazole S3307 (0.25 mg l⁻¹) and ABT root-promoting powder (0.5 mg l⁻¹) showed a significant improvement of hardening in regenerated plantlets, which were stronger and generated a better fibrous root system than the control. This improvement could facilitate the transgenic operation of maize.     

Ribosomal protein L6 promotes growth and cell cycle progression through upregulating cyclin E in gastric cancer cells

Our previous study revealed that human ribosomal protein L6 (RPL6) was upregulated in multidrug-resistant gastric cancer cells and over-expression of RPL6 could protect gastric cancer cells from drug-induced apoptosis. The present study was designed to explore the role of RPL6 in tumorigenesis and development of gastric cancer. The expression of RPL6 in gastric cancer tissues and normal gastric mucosa was evaluated by immunohistochemical staining. It was found RPL6 was expressed at a higher level in gastric cancer tissues than that in normal gastric mucosa. RPL6 was then genetically overexpressed or knocked down in human immortalized gastric mucosa epithelial GES cells. It was demonstrated that upregulation of RPL6 accelerated the growth and enhanced in vitro colony forming ability of GES cells whereas downregulation of RPL6 showed adverse effects. Moreover, over-expression of RPL6 could promote G1 to S phase transition of GES cells. It was further evidenced that upregulation of RPL6 resulted in elevated cyclin E expression while downregulation of RPL6 caused decreased cyclin E expression in GES cells. Taken together, these data indicated that RPL6 was overexpressed in human gastric cancer and its over-expression could promote cell growth and cell cycle progression at least through upregulating cyclin E expression.     

Pontine norepinephrine defects in Mecp2-null mice involve deficient expression of dopamine β-hydroxylase but not a loss of catecholaminergic neurons

Rett syndrome is a neurodevelopmental disorder caused by Mecp2 gene mutations. In RTT patients and Mecp2-null (Mecp2^−/Y) mice, norepinephrine (NE) content drops significantly, which may play a role in breathing arrhythmia, sleep disorders and sudden death. However, the underlying mechanisms for the NE defect are not fully understood. The NE defect may result from decreased NE biosynthesis, loss of catecholaminergic neurons or both. Although deficiency in tyrosine hydroxylase (TH) has been demonstrated, it is possible that dopamine β-hydroxylase (DBH), the critical enzyme converting dopamine to NE, is also affected. To test these possibilities, we studied DBH expressions in pontine catecholaminergic neurons of Mecp2^−/Y mice identified with breathing abnormalities. In comparison to the wild type, Mecp2^−/Y mice at 2 months of age showed ∼50% decrease in the expressions of DBH and TH, at both protein and mRNA levels in the locus coeruleus (LC) region. Consistently, DBH and TH immunoreactivity was markedly decreased in LC neurons of Mecp2^−/Y mice. No evidence was found for selective deficiency in TH- or DBH-containing neurons in Mecp2^−/Y mice, as almost all TH-positive cells expressed DBH. By counting TH-immunoreactive cells in the LC, we found that the Mecp2^−/Y mice lost only ∼5% of the catecholaminergic neurons as compared to wild-type, although their LC volume shrank by ∼15%. These results strongly suggest that the NE defect in Mecp2^−/Y mice is likely to result from deficient expression of not only TH but also DBH without significant loss of catecholaminergic neurons in the LC.