大豆品种合丰25体细胞胚发生条件的优化

为优化大豆体细胞胚再生植株的条件,提高大豆遗传转化的效率,本研究选用大豆品种合丰25为试验材料,针对影响体细胞胚诱导的3个关键因素pH值、2,4-D浓度及光照条件进行体细胞胚发生频率的研究。结果表明:pH值、2,4-D浓度、光照条件这3种因素的不同处理对体细胞胚的诱导影响显著,其幼胚的发生频率最高为70.70%、最低为3.70%。当pH值为7.0、2,4-D浓度为20mg/L,黑暗培养最有利于合丰25体细胞胚的诱导,其胚的发生频率为70.70%,明显高于其他处理。     

龙芽楤木愈伤组织诱导及胚状体发生初探

以楤木幼茎及根为试材,添加不同种类和浓度的激素,进行愈伤组织诱导、胚状体发生及幼苗分化。结果显示,幼茎在MS+2,4-D1.0mg/L培养基上愈伤组织诱导率最高。愈伤组织在MS+BA1.0 mg/L + NAA0.04 mg/L培养基上胚状体形成最多,幼苗分化率达92.5%,并能保持旺盛的再生能力。     

豆科牧草沙打旺抗乙硫氨酸变异系筛选

以NaN3诱变处理的沙打旺胚性愈伤组织为材料,用甲硫氨酸类似物—乙硫 氨酸为选择剂,筛选到1株抗性稳定且能再生植株的抗0.6 mmol L－1乙硫氨酸 变异系。该变异系细胞对乙硫氨酸的抗性是野生型细胞的8倍。当把变异系愈伤组织传入 体细胞 胚胎诱导培养基上,平均体细胞胚胎发生频率为13.9%,平均每g鲜重愈伤组织可产生21 个体     

大豆幼胚子叶体细胞胚诱导主要影响因素的研究

本研究选用东北地区4个主栽大豆品种的幼胚子叶, 探讨了培养基中氮源、生长素和蔗糖, pH值和低温预处理对体细胞胚诱导的影响; 并首次将低温预处理和pH=7.0的培养基应用到体细胞胚诱导中. 结果表明, MSB培养基附加40 mg/L 2,4-D和6%蔗糖, 其体细胞胚诱导率和诱导效率最高, 分别为49%和2.94%. 于4℃低温预处理的材料, 在pH=7.0     

掌叶半夏悬浮培养下的体细胞胚胎发生的研究

掌叶半夏种子在附加２，４－Ｄ２．０，ＢＡ０．５ｍｇ／Ｌ的ＭＳ培养基上形成浅黄色或白色颗粒状胚性愈伤组织。胚性愈伤组织在附加２，４－Ｄ１．０，ＢＡ０．５，ＣＨ３００ｍｇ／Ｌ的ＭＳ液体培养其中振荡培养，可产生大量的体细胞胚。２，４－Ｄ对体胚诱导效果显著并促进其早期发育，但抑制其进一步发育成熟。ＮＡＡ对体胚诱导效果不如２，４－Ｄ，但可使体胚正常发育。水解酷蛋白明显提高体胚诱导频率。显微观察表明：体胚起源     

锌指核酸酶(ZFN)介导的牛肌肉抑制素基因(MSTN)突变的研究

肌肉生成抑制素(myostatin,MSTN)是肌肉发育的负调控基因,突变后会引起肌肉的过度发育,在肉用动物育种中有非常重要的价值。本研究利用锌指核酸酶(zinc finger nuclease,ZFN)对牛(Bos taurus)MSTN基因的第二外显子特异性剪切,诱导该基因的突变,从而达到敲除该基因的目的。首先对脂质体介导的双质粒共转染牛成纤维细胞的条件进行优化,获得的共转染效率高达19.27%;之后利用两组ZFNs对牛成纤维细胞进行转染,单细胞通过口吸法移入96孔板,经扩大培养后进行PCR筛选,获得18株MSTN基因突变细胞株,其中包括一株双等位基因敲除的细胞系,两组ZFNs对牛成纤维细胞的突变效率分别为6.05%和3.84%;最后,挑选一株突变细胞系进行体细胞核移植研究,共获得24枚重构胚;将去除透明带的重构胚培养于2i培养液中,获得囊胚来源的类滋养层干细胞,通过PCR和测序检测,证明该细胞系在MSTN基因第二外显子具有与供体细胞相同的突变。结果表明,所合成的两组ZFNs可以在MSTN作用位点引起突变,具有很高的突变效率,而且能获得双等位基因突变的细胞株,获得的细胞株能够用作体细胞核移植的供体细胞。本研究为肉用牛的品质改良和育种工作提供了基础资料。     

CRISPR/Cas9介导的ASGR1基因敲除猪制备

猪(Sus scrofa)内皮细胞的去唾液酸糖蛋白受体1(asialoglycoprotein receptor 1,ASGR1)是诱发异种肝移植后受体发生血小板减少症的主要诱因之一.本研究在α-1,3-半乳糖基转移酶基因(α-1,3-galactosyltransferase,GGTA1)敲除的五指山小型猪基础上,利用规律成簇间隔短回文重复(clustered regularly interspaced short palindromic repeats/Cas9,CRISPR/Cas9)结合体细胞核移植技术制备ASGR1基因敲除猪,针对猪ASGR1基因设计并构建了靶向表达载体单链导向RNAl (single guide RNA1,sgRNA1),将sgRNA1与增强绿色荧光蛋白(enhanced green fluorescent protein,EGFP)质粒共同电转染GGTA1基因敲除猪耳成纤维细胞,流式细胞仪富集筛选带绿色荧光的细胞后培养单细胞克隆.实验结果表明,PCR测序检测培养获得的41个单细胞克隆,37个克隆发生基因突变,ASGR1基因突变效率为90％,其中双等位基因敲除的细胞克隆30个,效率高达73％.选择4个ASGR1基因敲除类型不同的细胞为核供体进行核移植,将早期重构胚移植到4头受体猪,2头妊娠至终期,产仔猪6头,Western blot显示ASGR1基因敲除仔猪的肝脏中没有ASGR1的表达.对sgRNA1的13个潜在脱靶位点,分别设计引物进行PCR扩增和测序,结果显示没有脱靶现象.总之,本研究利用CRISPR/Cas9结合体细胞核移植技术快速高效地制备了GGTA1/ASGR1基因敲除五指山小型猪模型,有望解决异种肝移植后出现的血小板减少症,为临床过渡肝的应用研究提供了良好的研究材料.     

3份长穗偃麦草种质的体细胞核型分析

采用根尖压片法,对不同国家来源的3份长穗偃麦草(Elytrigia elongata)种质材料的体细胞染色体核型进行分析,旨在为其细胞学特性和系统演化的研究奠定科学基础.结果表明:来源于中国新疆的EE001细胞染色体相对长度组成为1L+ 17M2+ 15M1+ 2S,核型公式为K(2n)=10X=70=50m+ 16sm+ 4st;来源于德国的EE014细胞染色体相对长度组成为3L+ 14M2 +13M1 +5S,核型公式为K(2n)=10X=70=48m+20sm+2st;来源于葡萄牙的EE020细胞染色体相对长度组成为5L+ 11M2 +15M1 +4S,核型公式为K(2n)=10X=70=38m+ 22sm+ 8st+2t.3份长穗偃麦草种质的体细胞染色体核型均为“2B”类型.     

Effect of somatic cell count on milk and protein yields and female fertility in Tunisian Holstein dairy cows

The effects of test-day somatic cell scores (SCS) variations on milk and protein yields, and calving to first service and calving to conception intervals were studied in Tunisian Holsteins. There were 34,129, 25,700, and 18,077 test-day production records collected on first, second, and third parity cows, respectively. Records were of cows calving between 1996 and 2004 in 160 herds. Somatic cell scores and milk and protein yields were analysed using a linear model that included herd-test-day date and herd–year interactions, calving season, calving age, and calving to conception interval. Reproductive trait model included herd–year interaction, calving season, calving age, and month of insemination. Effects of SCS on milk and protein yields were studied by regressing current test-day yields on corresponding and preceding test-day SCS, while effects of SCS on fertility traits were investigated by separately regressing calving to first service and calving to conception intervals corrected for environmental and management factors on SCS corrected for actual milk yield. A cow produced around 19.0 kg (SD = 8.0 kg) and 0.6 kg (SD = 0.3 kg) milk and protein yields on a daily basis and had an average of 3.8 (SD = 2.1) SCS in the first three lactations. SCS varied consistently (p < 0.05) with herd-test-day date and herd–year interactions in all lactations. Days in milk, calving age, and calving to conception interval were all together important sources of variation (p < 0.05) for SCS mainly in the first and second parities. Test-day milk and protein yields were unfavourably affected by high SCS recorded in the same test-day and with a lesser degree by SCS observed in the nearest preceding test-day. Reduction in milk and protein productions from increased SCS varied from 0.23 to 1.76 kg and from 6 to 75 g, respectively. Likewise, increased test-day SCS lengthened both calving to first service (mean interval = 94.9 days; SD = 49.1 days) and calving to conception (mean interval = 161 days; SD = 69.6 days) intervals by 1.3 to 2.0 days for each unit increase in SCS. Using SCS in addition to milk traits as a criterion to select semen and improving veterinary care should result in increased milk and protein yields and in satisfactory fertility measures.