Identification of quantitative trait loci for drought tolerance at seedling stage by screening a large number of introgression lines in maize

The maize genome hosts tremendous phenotypic and molecular diversity. Introgression lines (ILs), developed by continuous backcrossing to recurrent parents, could provide a unique genetic stock for quantitative trait locus (QTL) mapping. Using maize lines from six heterotic groups of different ecological zones, we developed >500 BC₂F₂ IL sets by crossing 11 inbred lines (as recurrent parents) with >200 local maize inbred lines (as donor parents). Of them, 34 IL sets were selected as a subset for drought tolerance screening and a total of 417 ILs survived under severe water stress at seedling stage. One set of 32 surviving ILs, derived from Chang7-2/DHuang212, was used for QTL mapping with simple sequence repeat markers covering the whole genome, with seven QTL detected. Furthermore, investigating all surviving ILs, we identified two common regions in bin 3.04, corresponding to marker intervals bnlg1904–umc1772 and umc1223–bnlg1957, respectively, which shared high genetic variation in three IL sets. Our results indicated that selective genotyping can be used to identify genetic loci for complex traits. The ILs, highly selected for drought tolerance in this study, provide a unique set of materials for both genomic studies and development of enhanced germplasm resources.     

鸡白细胞介素-2 cDNA克隆

用哺乳动物细胞表达克隆系统克隆了鸡ＩＬ－２ｃＤＮＡ。以ＣｏｎＡ（２０μｇ／ｍＬ）诱导ＳＰＦ鸡脾脏淋巴细胞,第２０小时收集细胞,提取ｍＲＮＡ,以ＳｕｐｅｒＳｃｒｉｐｔＰｌａｓｍｉｄＳｙｓｔｅｍｆｏｒｃＤＮＡＳｙｎｔｈｅｓｉｓ试剂盒合成ｃＤＮＡ,定向连结于穿梭质粒ｐＳＶ·ＳｐｏｒｔⅠ,构建ｃＤＮＡ文库。将文库分组提取重组质粒,转染ＣＯＳ－７细胞,表达ｃＤＮＡｓ。以淋巴细胞增殖试验检测表达产物ＩＬ－２活性。经过３轮筛选,获得１４个具有ＩＬ－２活性的克隆。选择其中４个进行酶切分析,结果显示这４个ｃＤＮＡ大小分别为１．５,１．０,０．８和０．８ｋｂ。     

猪IL-6 cDNA的克隆及在大肠杆菌中的高效表达

运用RT—PCR方法,从经刀豆球蛋白A(ConA)诱导的猪外周血单核细胞(PMBcs)总RNA中扩增得到了猪IL-6(porcine IL-6,pIL-6)的cDNA,并将其克隆到pGEM—T载体上。DNA序列测定表明,克隆得到的pIL-6cDNA与国外报道的完全一致,包括终止密码子在内其编码区的长度为639bp,编码212个氨基酸残基的蛋白质。将pIL-6成熟肽段编码区亚克隆至pET-28(a)中,构建成原核表达质粒pETPIL6。该质粒的BL21(DE3)LysS转化菌在IPTG的诱导下可高效表达plL-6,表达量占菌体总蛋白的30．60%-38．35％。     

制备性分段电泳纯化基因工程白细胞介素—2

采用具有高分辨率特性的聚丙烯酰胺凝胶为载体,以电泳技术将工程菌株的各种蛋白成分精细分离,再用紫外检测手段对白细胞介素—2(IL—2)基因表达产物在凝胶上定位,通过准确地切割凝胶,提取其中的蛋白质,获得了高纯度(>95%)的基因工程IL—2产物.经用同位素掺入法测定,其对小鼠胸腺细胞有明显的促进增殖活性     

串联IL-2的犬瘟热病毒F-H融合基因重组乳酸杆菌的构建与表达

为了研发犬瘟热乳酸杆菌载体疫苗,更好地预防犬瘟热,试验采用重叠延伸PCR(SOEPCR)技术扩增IL2-F-H融合基因,将IL2-F-H融合基因亚克隆至穿梭载体pSIP409上,构建pSIP-IL2-F-H重组表达载体,并电转化至植物乳杆菌NC8感受态细胞中,构建串联水貂白细胞介素-2(IL-2)的犬瘟热病毒(CDV)F-H融合基因重组乳酸杆菌;利用SDS-PAGE和Western-blot检测IL2-F-H融合蛋白的表达情况。检测表明,重组乳酸杆菌表达了分子量约为80.16kDa的IL2-F-H融合蛋白,且该融合蛋白能与CDV抗体发生反应,具有反应原性。     

雏鸡感染网状内皮组织增殖病病毒后免疫病理的研究

 研究了 1日龄 SPF雏鸡感染网状内皮组织增殖病病毒 ( REV)后 ,胸腺和脾脏 T细胞增殖反应及 T细胞数量、法氏囊和脾脏 B细胞增殖反应和抗体生成细胞数量、胸腺和脾脏 IL- 2及 IFN诱生活性的动态变化。结果发现 ,雏鸡感染 REV后 ,胸腺和脾脏 T细胞增殖反应及数量、法氏囊和脾脏 B细胞增殖反应和 Ig G+、Ig M+、Ig A+抗体生成细胞数量均明显低于未感染对照雏鸡 ;胸腺和脾脏 IL- 2及脾脏 IFN诱生活性显著下降 ,表明感染 REV后雏鸡中枢和外周免疫器官细胞免疫和体液免疫降低及细胞因子 IL- 2及 IFN免疫调节减弱。