High-level expression of a novel recombinant human plasminogen activator (rhPA) in the milk of transgenic rabbits and its thrombolytic bioactivity in vitro

The human tissue-type plasminogen activator (tPA) is a key kinase of fibrinolysis that plays an important role in dissolving fibrin clots to promote thrombolysis. The recombinant human plasminogen activator (rhPA) has more thrombolytic advantages than the wild type tPA. To increase the half-life and thrombolytic activity of tPA, a mutant containing only the essential K2 fibrin-binding and P activating plasminogen domains of the wild type tPA was cloned. This fragment was then inserted into goat β-casein regulatory sequences. Then, a mammary gland-specific expression vector, PCL25/rhPA, was constructed, and the transgenic rabbits were generated. In this study, 18 live transgenic founders (12♀, 6♂) were generated using pronuclear microinjection. Six transgenic rabbits were obtained, and the expression levels of rhPA in the milk had a range of 15.2–630 µg/ml. A fibrin agarose plate assay of rhPA showed that it had strong thrombolytic bioactivity in vitro, and the highest specific activity was >360 (360 times more than that of alteplase). The results indicated that the rhPA containing only the K2 and P domains is efficiently expressed with higher thrombolytic bioactivity in the milk of transgenic rabbits. Our study also demonstrated a new method for the large-scale production of clinically relevant recombinant pharmaceutical proteins in the mammary glands of transgenic rabbits.     

Precise mapping Fhb5, a major QTL conditioning resistance to Fusarium infection in bread wheat (Triticum aestivum L.)

Qfhi.nau-5A is a major quantitative trait locus (QTL) against Fusarium graminearum infection in the resistant wheat germplasm Wangshuibai. Genetic analysis using BC(3)F(2) and BC(4)F(2) populations, derived from selfing two near-isogenic lines (NIL) heterozygous at Qfhi.nau-5A that were developed, respectively, with Mianyang 99-323 and PH691 as the recurrent parent, showed that Qfhi.nau-5A inherited like a single dominant gene. This QTL was thus designated as Fhb5. To fine map it, these two backcross populations and a recombinant inbred line (RIL) population derived from Nanda2419?×?Wangshuibai were screened for recombinants occurring between its two flanking markers Xbarc56 and Xbarc100. Nineteen NIL recombinants were identified from the two backcross populations and nine from the RIL population. In the RIL recombinant selection process, selection against Fhb4 present in the RIL population was incorporated. Genotyping these recombinant lines with ten markers mapping to the Xbarc56-Xbarc100 interval revealed four types of Mianyang 99-323-derived NIL recombinants, three types of PH691-derived NIL recombinants, and four types of RIL recombinants. In different field trials, the percentage of infected spikes of these lines displayed a distinct two-peak distribution. The more resistant class had over 55% less infection than the susceptible class. Common to these resistant genotypes, the 0.3-cM interval flanked by Xgwm304 and Xgwm415 or one of these two loci was derived from Wangshuibai, while none of the susceptible recombinants had Wangshuibai chromatin in this interval. This interval harboring Fhb5 was mapped to the pericentromeric C-5AS3-0.75 bin through deletion bin mapping. The precise localization of Fhb5 will facilitate its utilization in marker-assisted wheat breeding programs.     

温度对椰心叶甲实验种群生长的影响

在不同温度(16、20、2、28和32 ℃)条件下观察椰心叶甲的发育、存活及繁殖情况.结果表明,椰心叶甲在32 ℃恒温下不能存活,在16～28 ℃世代发育起点温度为11.08 ℃,有效积温为966.22日·度,在海南省儋州市1年可发生4～5代.椰心叶甲世代存活率在28 ℃时最高,达92.5%.净增殖率、内禀增长率及周限增长率均在28 ℃时最高,分别为54.1、0.0260和1.0263.平均世代历期和种群加倍时间分别以20 ℃和28 ℃时最短,前者为123.1 d,后者为26.7 d.种群趋势指数以28 ℃时最高,为50.8,表明24～28 ℃为椰心叶甲种群生长的适合温度.     

Purification and characterization of a novel chitinase gene from Paecilomyces thermophila expressed in Escherichia coli

A novel chitinase gene (PtChiA) from the thermophilic fungus Paecilomyces thermophila was cloned and expressed in Escherichia coli as an intracellular soluble protein. The gene sequence alignment indicates that PtChiA belongs to glycoside hydrolase (GH) family 18 and has an open reading frame comprising of 1473 bp nucleotide sequences with five introns. PtChiA encodes 400 amino acids without any predicted signal peptide. PtChiA was purified by Ni-IDA chromatography. It displayed an acidic optimum pH of 4.5 and broad pH stability (pH 4.0-10.5). The enzyme exhibited an optimal temperature of 50°C and was stable up to 40°C. PtChiA was strongly inhibited by anionic detergent SDS, and also by metal ions Hg(2+) and Mn(2+). It did not exhibit any antifungal activity against pathogenic fungi. It has the ability to hydrolyze colloidal chitin into chito-oligomers suggesting its use in conversion of chitin waste into chito-oligosaccharides.     

pmX: a recessive powdery mildew resistance gene at the Pm4 locus identified in wheat landrace Xiaohongpi

Powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is one of the most devastating foliar diseases of wheat and imposes a constant challenge on wheat breeders. Xiaohongpi, a Chinese landrace of wheat (Triticum aestivum L.), shows resistance to powdery mildew during the entire growth stage in the field and under controlled conditions. The F₁ plants from cross of the powdery mildew susceptible cultivar Yangmai158 with Xiaohongpi were susceptible to isolate Bgt19, the locally most prevalent Bgt isolate. In the derived F₂ population and F₃ progenies, the resistance segregation deviated significantly from the one-gene Mendelian ratio. However, marker analysis indicated that only one recessive gene conferred the resistance, which co-segregated with Xsts-bcd1231 that showed co-segregation with Pm4a in different studies. Allelism test indicated that this recessive resistance gene, designated as pmX, is either allelic or tightly linked to Pm4a. The pmX gene was different from Pm4 alleles in resistance spectrum. Examination of the genotype frequencies at pmX and the linked marker loci in the F₂ population showed that a genetic variation favoring the transmission of Xiaohongpi alleles could be the cause of deviated segregation. Mapping of the pmX-linked markers using Chinese Spring deletion lines indicated that it resides in the 0.85–1.00 bin of chromosome 2AL.     

Association of jacalin-related lectins with wheat responses to stresses revealed by transcriptional profiling

Jacalin-related lectins (JRLs) are carbohydrate-binding proteins widely present in plants and have one or more jacalin domains in common. However, JRLs’ structural types and functions are still poorly understood. In the present study, a total of 67 wheat (Triticum aestivum) JRL genes were identified through an exhausted search of EST database coupling with genome walking using published 454 sequence reads of Chinese Spring. A comparison of the translated wheat JRL proteins with those from other plants showed plant JRLs generally had low sequence similarity within and between species but exhibited conserved modular domain structures. More JRL genes encoded multiple jacalin domains in Arabidopsis thaliana, whereas more genes encoded chimeric JRLs in cereal plants. Dirigent domain-containing JRL genes were Poaceae-specific and accounted for nearly half of the identified wheat JRL genes. The dirigent domains were evolutionarily significantly correlated with the covalently linked jacalin domains. A phylogenetic analysis showed JRL proteins have experienced a substantial diversification after speciation. Moreover, new structural features conserved across the taxa were identified. Digital expression analysis and RT-PCR assays showed the expression of wheat JRL genes was largely tissue specific, typically low, and mostly inducible by biotic and abiotic stresses and stress hormones. These results suggest plant JRLs are critical for plant adaptation to stressful environments.     

林麝幼体的换毛序和胎毛、夏毛、冬毛的形态

被毛是哺乳动物最主要的特征之一,有关动物换毛序与环境关系的研究,是动物生态学研究的一个重要方面。从1853年Browne开始研究毛发结构以来,人们对毛发的形态学、分类学和种属关系等方面的研究已开展了大量工作,特别是随着电镜技术的应用,这些方面的工作在逐渐深入(曹汉民等,19     

Biological assessment of Tetrastichus brontispae,a pupal parasitoid of coconut leaf beetle Brontispa longissima

To control coconut leaf beetle, Brontispa longissima (Gestro), the pupal parasitoid Tetrastichus brontispae Ferrière was imported from Taiwan and its biology was studied in quarantine in Hainan, China. The parasitoid development includes an egg, three larval instars and three pupal stages. Its developmental time from egg to adult was 19.5±0.5 days under conditions of 24±2°C and 75±5% relative humidity (RH). Temperature had no effect on the sex ratio of offspring, but significantly affected the parasitism rate and reproduction. The parasitism rates were 98.07, 97.97 and 95.03% at 28, 24 and 20°C, respectively, whereas the parasitism rate was 52.18% at 18°C and 69.48% at 30°C, respectively. Furthermore, the parasitoids reared at 18 and 30°C produced fewer offspring than those at 20, 24 and 28°C, respectively. With the increase in temperature, developmental time decreased linearly from 46.19 days at 18°C to 17.10 days at 28°C. RH significantly influenced development, parasitism rate and the reproduction of T. brontispa. With the decrease of RH, developmental time increased from 22.94 days at 20% RH to 18.84 days at 95% RH. In contrast, parasitism rate and the number of offspring per female increased with the increase of RH. Though emergence rates between 50 and 95% RH were much higher than those between 20 and 35% RH, the sex ratios between 20 and 95% RH were not different. Photoperiod had no effect on parasitism, the number of offspring per female, emergence and the sex ratio of T. brontispae, but developmental time was significantly different for different photoperiods. Sucrose, honey and glucose significantly enhanced adult longevity, parasitism and the number of offspring per female of T. brontispae, but had no effect on the sex ratio and survival. Females of T. brontispae only parasitized fourth to fifth larval instars and 1–5-day-old pupae, but there was a significant difference in the number of offspring per female, development time, emergence and the sex ratio of offspring in different instars. These results showed that 1-day-old pupae, a temperature of 24–28°C and 65–95% RH were optimal for T. brontispae. These findings should be helpful in developing a production system to rear and release T. brontispae in large enough quantities to effectively control coconut leaf beetle.     

Mapping QTLs controlling kernel dimensions in a wheat inter-varietal RIL mapping population

Key message

Seven kernel dimension QTLs were identified in wheat, and kernel thickness was found to be the most important dimension for grain weight improvement.

Abstract

Kernel morphology and weight of wheat (Triticum aestivum L.) affect both yield and quality; however, the genetic basis of these traits and their interactions has not been fully understood. In this study, to investigate the genetic factors affecting kernel morphology and the association of kernel morphology traits with kernel weight, kernel length (KL), width (KW) and thickness (KT) were evaluated, together with hundred-grain weight (HGW), in a recombinant inbred line population derived from Nanda2419?×?Wangshuibai, with data from five trials (two different locations over 3 years). The results showed that HGW was more closely correlated with KT and KW than with KL. A whole genome scan revealed four QTLs for KL, one for KW and two for KT, distributed on five different chromosomes. Of them, QKl.nau-2D for KL, and QKt.nau-4B and QKt.nau-5A for KT were newly identified major QTLs for the respective traits, explaining up to 32.6 and 41.5% of the phenotypic variations, respectively. Increase of KW and KT and reduction of KL/KT and KW/KT ratios always resulted in significant higher grain weight. Lines combining the Nanda 2419 alleles of the 4B and 5A intervals had wider, thicker, rounder kernels and a 14% higher grain weight in the genotype-based analysis. A strong, negative linear relationship of the KW/KT ratio with grain weight was observed. It thus appears that kernel thickness is the most important kernel dimension factor in wheat improvement for higher yield. Mapping and marker identification of the kernel dimension-related QTLs definitely help realize the breeding goals.