Relationship of p21WAF1 gene polymorphisms with protein expression in breast carcinomas

AIM: To investigate the relationship between p21^WAF1gene polymorphisms and protein expression in breast carcinoma. METHODS: Polymerase chain reaction single-strand conformation polymorphisms technique (PCR-SSCP) and immunohistochemical assay of S-P immunostaining technique were used to study polymorphisms of p21^WAF1 and protein expression respectively on the specimen of paraffin-embedded tissues in 100 cases of breast carcinomas and 40 benign breast diseases as control. RESULTS: Two p21^WAF1 gene polymorphisms were found in 18% (18/100) of breast carcinomas and 5% (2/40) of control samples. The difference between the two groups was statistically significant (χ²=3.94, P＜0.05). The positive immunohistochemical reaction of p21^WAF1 protein were found in 50% (50/100) of breast carcinomas and 12.5% (5/40) of control samples. The difference between the two groups was statistically significant (χ²=16.84, P＜0.01). The positive immunohistochemical reaction of p21^WAF1 protein were found in 100% (18/18) of breast carcinomas with p21^WAF1 gene polymorphisms and 39% (32/82) of no p21^WAF1 gene polymorphisms. The difference between two groups was statistically significant (χ²=21.95, P＜0.01). The p21^WAF1 gene polymorphisms were correlated with the protein expression in breast carcinomas (r=0.576, P＜0.01). CONCLUSION: p21^WAF1 gene polymorphisms may create the different copies of mRNA and may make relevant protein molecules.     

禽传染性喉气管炎病毒体内外感染模型中鸡Toll样受体21mRNA转录水平的研究

为研究病毒与机体的相互作用,本研究参考GenBank中鸡Toll样受体21 (ChTLR21)的基因序列设计实时定量PCR特异性引物,以鸡核糖体蛋白L4 (RPL4)为内参基因,建立检测ChTLR21 mRNA相对转录水平的实时定量PCR方法,分析ChTLR21在禽传染性喉气管炎病毒(ILTV)感染的鸡胚成纤维细胞(CEF)中和感染SPF雏鸡免疫器官脾脏、法氏囊和胸腺组织中的转录水平.结果显示:ILTV感染CEF后2h、4h、8h和18h时间点ChTLR21 mRNA转录水平分别为未感染对照细胞的1 540.53、0.98、1.19和3.70倍.但仅在ILTV感染2h时引起ChTLR21转录水平升高,与对照组相比差异显著(P＜0.05).ILTV感染SPF雏鸡后6h、24 h和30 h脾脏中ChTLR21 mRNA转录水平分别为未感染对照的56.34、59.85和3.61倍;法氏囊中分别为0.03、25.98和3.08倍;胸腺中分别为2.52、50和7.32倍.在感染初期,脾脏中ChTLR21转录量显著升高,随后有所降低,但均高于未感染对照(p＜0.05);法氏囊中仅在感染6h时呈显著抑制(p＜0.01);胸腺中呈波动性转录水平升高,但与对照组无统计学差异(p＞0.05).本研究证明ChTLR21参与了鸡体对ILTV感染的应答,并在体内外感染模型中呈现不同的表达规律.     

Reduction in cadmium accumulation in japonica rice grains by CRISPR/Cas9-mediated editing of OsNRAMP5

Cadmium (Cd) intake is harmful to human health and Cd contamination in rice grains represents a severe threat to those consuming rice as a staple food. Knockout of Cd transporters is a promising strategy to reduce Cd accumulation in rice grains. OsNRAMP5 is the major transporter for Cd and manganese (Mn) uptake in rice. Nevertheless, it is uncertain whether knockout of OsNRAMP5 is applicable to produce low Cd rice without affecting plant growth and grain yield. In this study, we adopted CRISPR/Cas9-based gene editing technology to knock out OsNRAMP5 in two japonica varieties. We generated three independent transgene-free osnramp5 mutants and investigated the effect of osnramp5 mutations on Cd accumulation and plant growth. Hydroponic experiments showed that plant growth and chlorophyll content were significantly reduced in osnramp5 mutants at low Mn conditions, and this defective growth in the mutants could be fully rescued by supply of high levels of Mn. Cd and Mn accumulation in both roots and shoots was markedly reduced in the mutants compared to that in wild-type plants. In paddy field experiments, although Cd in flag leaves and grains was greatly reduced in osnramp5 mutants, some agronomic traits including plant height, seed setting rate, and grain number per panicle were affected in the mutants, which ultimately caused a mild reduction in grain yield. The reduced plant growth in the mutants can be attributed to a marked decrease in Mn accumulation. Our results reveal that the manipulation of OsNRAMP5 should be treated with caution: When assessing the applicability of osnramp5 mutants, soil pH and soil water content in paddy fields need to be taken into consideration, since they might affect the levels of available Mn in the soil and consequently determine the effect of the mutation on grain yield.     

转Xa21基因杂交水稻选育和评价

通过基因枪转基因方法和双质粒共转化体系将Xa21基因转入优良恢复系明恢63,得到转基因系M12和M22,并进一步做田间试验、新品种选育和食用安全性评价。结果显示:M12和M22对中国的所有白叶枯病小种都表现出高度抗性,但与原受体品种明恢63相比,农艺性状上有许多变异,主要表现在:结实率、千粒重等农艺性状变差,与珍汕97A的配合力显著降低;但与温敏不育系(X07S、056S)配组的F1有较好的田间表现。通过多代回交和分子标记辅助选择,成功地将Xa21基因从M12株系转到保持系80-4B和不育系80-4A中,得到抗白叶枯病的皖21B和皖21A。并利用皖21A不育系选育出优良杂交组合抗优97(皖21A×R-18),该组合在两年区域试验和一年生产试验中产量表现突出,米质优良。对不同世代和不同遗传背景的转基因品系进行白叶枯病鉴定和Southern分析表明:Xa21基因都能稳定遗传和正常表达,连续16代的种植并没使其白叶枯病的抗性减弱或丧失,而且不论Xa21基因是纯合的还是杂合的都有相同的抗性表现。对大鼠和小鼠的饲喂试验表明:转基因大米实质上等同于非转基因大米,是安全无害的。     

人工合成抗菌肽D2A21基因的克隆和表达

为了获得高效表达的抗菌肽D2A21,本研究根据抗菌肽D2A21的氨基酸序列及大肠杆菌偏爱的密码子,设计并人工合成D2A21基因,并将该合成基因克隆至pProEXHTb载体中,构建成含有含6个组氨酸标签的重组质粒。重组质粒转化至大肠杆菌中,经摇瓶发酵,IPTG诱导后,发酵液用聚丙烯酰胺凝胶电泳分析。结果表明,成功合成D2A21基因,并构建了抗菌肽D2A21表达载体。通过镍柱亲和层析纯化,聚丙烯酰胺凝胶电泳分析,抗菌肽D2A21能够在大肠杆菌中稳定表达。本研究将为大规模表达纯化D2A21及其进一步的研究和利用提供一定基础。     

METTL21C蛋白的序列特征、三维结构及分子进化

METTL21C蛋白作为甲基转移酶参与多种细胞进程的翻译后修饰,在生命活动中发挥重要的调控作用。从蛋白质信息数据库UniprotKB中选取36个不同代表物种的METTL21C蛋白作为研究对象,理化参数分析显示一级序列中氨基酸残基个数差异较大,平均为254个氨基酸残基,大多为偏酸亲水性蛋白。系统进化树显示36种METTL21C蛋白共分为哺乳类、爬行类、鸟类和鱼类4支,与物种的亲缘进化关系基本吻合。多序列比对和motif分析结果表明不同物种METTL21C蛋白的序列相似度较高,10个motif序列几乎覆盖了全长多肽链,大都含有motif 1～motif 5,但N端序列差异较大,在哺乳动物中包含特有的motif 7、motif 8和motif 10,而在多种鸟类中有大段序列缺失。结构对比分析发现不同物种METTL21C蛋白的空间构象绝大部分区域近乎完全重叠,仅少部分自由度较高的局部肽段有一定程度的差异,表明不同物种METTL21C在进化过程中一级序列有一定的差异,但是空间结构极为相似。以人METTL21C为蛋白质互作网络中心,直接相关蛋白质共11种,间接相关蛋白质共9种,说明METTL21C直接或间接参与多种细胞进程,调控机体的生命活动,为进一步深入研究METTL21C的生物学功能及其分子机制提供了理论基础。     

分子标记辅助选择改良杂交水稻的白叶枯病抗性

分子标记辅助选择技术以其对目标基因快速而精确的选择为回交育种提供了非常有效的工具。华中农业大学作物遗传改良实验室运用分子标记辅助的回交育种方法,将广谱高抗白叶枯病基因Ｚａ２１迅速导入到杂交水稻的两个优良恢复系明灰６３和６０７８之中,成功地完成了其抗生改良,并通过配组而达到了改良杂交水稻抗性的目的。本研究所运用的分子标记辅助的水稻回交育种程序是：通过一次杂交、二次回交改良杂交水稻抗性的目的。本研究所     

播期对洛麦21生长发育的影响

对洛麦21的生育规律进行了研究。结果表明:洛麦21的幼穗分化总体表现为前期慢、后期快,随着播期的推迟,幼穗分化的时间缩短。平均灌浆速率在1.453~1.697之间,在10月4日播种下平均灌浆速率最大,灌浆时间35~38 d,千粒重稳定在45 g以上。针对灌浆速率而言,以10月上旬适期播种对洛麦21最为有利。洛麦21主要依靠主茎和第1、2个一级分蘖成穗。随着播期的推迟,洛麦21的总分蘖数明显降低,成穗率提高。以10月14日播种的产量最高。     

利用Xa21基因改良杂交稻白叶枯病抗性

以转Xa 21基因的水稻优良恢复系BR527为抗白叶枯病基因供体,分别与不育系金23A、G46A、D62A、D200A进行组配,研究该基因在改良杂交水稻白叶枯病抗性方面的作用。分子检测表明,供体基因能够稳定遗传给杂交组合;田间接种鉴定发现杂交组合对P1,P2,P3,P4,P6和CII,CIV,CV等白叶枯病菌生理小种均表现出抗或高抗,揭示Xa 21基因的抗性好,抗谱广;农艺性状考察结果显示这些杂交组合的农艺性状与对照组合比较没有明显改变。说明该基因可以作为一个显性抗白叶枯病基因在杂交水稻抗性改良上上加以应用,与此同时,得到了四个白叶枯病抗性改良的优良杂交水稻组合。