昆虫遗传转化品系的常用标记

遗传转化标记是将遗传修饰昆虫从野生型种群中分辨出来的根据,遗传转化昆虫的鉴定、转化品系的维持及其遗传稳定性的监测都依赖于可靠的标记系统,发展易于应用和监测的转化标记能够极大地促进害虫遗传防治的相关研究。用于遗传修饰昆虫的转化标记主要有昆虫眼睛颜色标记基因、抗药性标记基因和荧光蛋白标记基因等。非果蝇类昆虫首个遗传转化品系的鉴定是通过眼睛颜色突变而实现,但大多数昆虫物种没有可用的突变体或缺少相应基因的信息,从而限制了眼睛颜色标记的应用。抗药性基因标记虽然能够通过对转化昆虫进行集体选择而大幅度提高筛选转化体的效率,但由于其鉴定的准确性不高且存在安全性问题,未得到广泛应用。荧光蛋白标记基因的发展则显著拓宽了能够转化的昆虫种类。从水母分离的绿色荧光蛋白(GFP)经突变方法获得了多种不同荧光性质的突变体,经人为修饰后与适宜的强启动子构成转化标记载体,能够有效鉴定更多昆虫物种的遗传转化个体,其中应用较多的是增强型绿色荧光蛋白(EGFP)。此外,从珊瑚属海葵中分离得到的红色DsRed标记基因提供了多样化的红色荧光蛋白选择,在某些生物中DsRed与GFP联合应用的表现明显优于GFP突变体,所以其应用前景也非常广泛。本文着重从眼睛颜色、抗药性和荧光蛋白等3个方面阐述了标记基因的发展历史与现状,并对其今后的发展方向进行了展望。

上皮间质转化在胃癌及相应转移淋巴结中的表达及意义

目的探讨上皮间质转化(EMT)标志物在胃癌组织和转移淋巴结中的表达情况及其与恶性生物学行为的相关性。方法采用免疫组化法检测145例胃癌、25例异常增生、13例肠化、42例转移淋巴结及40例正常胃黏膜组织中EMT标志物Ecadherin、β-catenin、N-cadherin、转化生长因子β1(TGF-β1)及Snail的表达情况。结果胃癌组织中E-cadherin、β-catenin、Ncadherin、TGF-β1及Snail的阳性表达率分别为73.5%、65.5%、14.5%、53.1%、35.9%,正常胃黏膜组织中分别为100%、100%、0%、27.5%、2.5%,差异有统计学意义(P<0.05)。E-cadherin、β-catenin表达水平的降低及TGF-β1表达水平的升高与胃癌浸润程度有关(P<0.05)。E-cadherin与β-catenin的表达呈正相关,而与TGF-β1呈负相关(P<0.05)。N-cadherin与TGF-β1的表达呈正相关(P<0.05)。转移淋巴结中,E-cadherin和β-catenin的表达水平高于胃癌组织,而TGF-β1的表达低于胃癌组织(P<0.05)。结论 TGF-β1和Snail的高表达以及E-cadherin、β-catenin、N-cadherin的低表达与胃癌的浸润和转移有密切关系;在胃癌的发展过程中,E-cadherin向N-cadherin转化及TGF-β1表达可能发挥重要作用。淋巴结转移灶中出现了间质上皮转化(MET)。

老年脑胶质瘤组织中骨膜蛋白及EMT标记物的表达及临床意义

目的探讨老年脑胶质瘤组织中骨膜蛋白(POSTN)及上皮-间质转化(EMT)标记物的表达及临床意义。方法回顾性收集完成神经外科手术治疗的105例老年脑胶质瘤患者临床资料,患者脑胶质瘤标本、正常组织标本均保存完整,比较脑胶质瘤组织和正常组织中POSTN及EMT标记物〔上皮钙黏蛋白(E-cad)、纤连蛋白(Fn)、波形蛋白(Vim)、神经钙黏蛋白(N-cad)〕的表达,比较低级别脑胶质瘤(LGG,Ⅰ级或Ⅱ级)与高级别脑胶质瘤(HGG,Ⅲ级或Ⅳ级)组织中POSTN及EMT标记物的表达,分析POSTN及EMT标记物的表达与老年脑胶质瘤相关性。结果脑胶质瘤组织中POSTN、Fn、Vim、N-cad表达高于正常组织,E-cad表达低于正常组织,差异有统计学意义(P<0.05);高级别脑胶质瘤组织中POSTN、Fn、Vim、N-cad表达高于低级别脑胶质瘤组织,E-cad表达低于低级别脑胶质瘤组织,差异有统计学意义(P<0.05);脑胶质瘤组织中POSTN、Fn、Vim、N-cad表达与脑胶质瘤恶性程度呈正相关(P<0.05);E-cad表达与脑胶质瘤恶性程度呈负相关(P<0.05);POSTN与Fn、Vim、N-cad表达呈正相关(P<0.05);POSTN与E-cad表达呈负相关(P<0.05)。结论老年脑胶质瘤的发生发展与组织中POSTN表达、EMT标记物有一定联系,检测POSTN及EMT标记物或可指导老年脑胶质瘤病情评估。

无选择标记的植物表达载体的构建

以双元载体pBINPLUS为基础,在T-DNA侧翼区通过2次特异PCR、酶切和连接相结合的方法,构建了1个无标记载体pBINMF(marker-free vector)。通过酶切后测序分析,这个无标记载体在T-DNA左、右边界之间只有1个多克隆位点(multiple cloned sites,MCS)。为了验证该载体的遗传稳定性,将gus基因克隆到该载体上并通过农杆菌介导的方法转化番茄栽培品种‘Moneymaker’,特异PCR扩增目的基因和GUS组织染色结果表明,gus基因已经整合进入番茄基因组并得以表达。该载体为今后直接获得无标记基因、生物安全的转化体,尤其可为马铃薯、木薯等无性繁殖材料的无标记基因转化提供可靠、有效的工具。

GFP标记的马铃薯大丽轮枝菌生物学特性研究

为了研究马铃薯黄萎病菌的侵染机制,将绿色荧光蛋白基因(GFP)通过农杆菌介导的遗传转化方法转入马铃薯黄萎病菌VD012中,经过潮霉素选择性培养基的筛选和分子鉴定,获得了47株有绿色荧光信号的阳性转化子。随机挑取8株阳性转化子,以野生型菌株为对照,对转化子的菌落形态、菌丝的生长速率、产孢量、粗毒素含量和致病力进行了研究。结果表明,8株阳性转化子中有3株微菌核产生的数量明显高于野生型,1株转化子微菌核产生量低于野生型菌株;各阳性转化子的生长速率和野生型菌株差异不显著;阳性转化子的产孢量与对照相比,有2株差异不显著。其余均有不同程度的降低。保湿培养8 h,所有阳性转化子的平均萌发率低于野生型菌株。相比对照,粗毒素的含量表现为升高趋势的转化子有7株,1株表现为下降趋势。致病力测定的结果表明,致病力增强的转化子有1株,2株转化子的致病力呈现降低的趋势。

显微CT对兔骨质疏松模型的实验研究

目的探讨去势法联合皮质激素快速诱导兔骨质疏松模型椎体骨小梁Micro-CT动态变化规律。方法 40只8个月龄雌性大白兔随机分为假手术组(A组,n=20)及去势+肌注甲强龙组(B组,n=20)。术后第2、4、8、10周行血清骨转化标志物检测及离体腰5椎体Micro-CT扫描。每组在每个时间点动物数5只。结果两组vBMD在术后第8周开始,差异有统计学意义,而BVF、Tb.N、Conn.D、Tb.Sp、SMI及Tb.Th分别在术后4、8周开始,差异有统计学意义。B组术后TRAP-5b迅速增高,至第4周达峰值,术后第4、8、10周,两组TRAP-5b差异有统计学意义;而BALP缓慢逐渐增高,至第8周才达峰值,术后第8、10周,两组BALP差异有统计学意义。结论去卵巢+肌注甲强龙可在8周内快速建立兔OP模型,骨微结构衰败早于vBMD变化而与骨转化标志物同步。

乳癌术后方对HER2过表达乳腺癌小鼠复发转移中HER2信号传导通路及相关基质蛋白表达的影响

目的探讨乳癌术后方对HER2介导的p38 MAPK、PI3K-Akt信号通路及其对HER2过表达乳腺癌细胞运动和黏附能力的影响,评价乳癌术后方抗HER2过表达乳腺癌复发转移的作用机制。方法选取30周龄左右发瘤相当的HER2/neu转基因自发乳腺癌小鼠,行原发肿瘤切除术,术后随机分为对照组、中药组(乳癌术后方组)、西药组(曲妥珠单抗组)、联合组(乳癌术后方+曲妥珠单抗组),干预4个月。评价肿瘤的复发率、瘤积抑制率及肺转移抑制率;并采用Western blot法检测干预结束后各组小鼠复发肿瘤组织中HER2相关信号通路中Total-Akt、p-Akt、p38、p-p38的表达及与细胞运动和黏附相关的基质蛋白细胞间黏附分子(E-cadherin)、上皮间质转化标记物波形蛋白(Vimentin)的表达情况。结果①各组小鼠肿瘤复发率差异无统计学意义(P>0.05)。对照组治疗结束时平均瘤积为11.11 cm3,中药组为5.56 cm3,组间差异有统计学意义(P=0.037);对照组平均肺转移结节数为16个,中药组为10个,中药组肺转移抑制率达37.85%,但组内差异无统计学意义(P>0.05)。②中药组、西药组、联合组p-p38及p-Akt表达含量较对照组明显下调(P<0.05);对照组与联合组E-cadherin几乎不表达,中药组与西药组E-cadherin表达显著增加(P<0.05)。各组复发肿瘤组织中Vimentin表达水平相似。结论乳癌术后方能够抑制HER-2/neu转基因小鼠术后乳腺复发肿瘤瘤积的增长,阻断HER2介导的p38 MAPK及PI3K-Akt信号通路,抑制肿瘤细胞增殖,促进肿瘤细胞凋亡,发挥抗肿瘤作用;乳癌术后方能够上调细胞间黏附分子E-cadherin的表达,其可能通过增加细胞间的黏聚力,维持乳腺上皮细胞的完整性,发挥抗复发转移作用。

Physiological Character and Gene Mapping in a New Green-revertible Albino Mutant in Rice

A green-revertible albino mutant-Qiufeng M was found from the japonica rice （Oryza sativa L. ssp. japonica） Qiufeng in the field. The first three leaves of the mutant were albino with some green. The leaf color became pale green since the fourth leaf and the glume had the same phenomenon as the first three leaves. The measuring data of the pigment content confirmed the visually observed results. It truly had a remarkable changing process in the leaf color in Qiufeng M. Comparison of the main agronomic characters between Qiufeng and Qiufeng M indicated that the neck length and grain weight showed significant difference at the 1% level, and other characters were not different. Genetic analysis showed that the green-revertible albino trait was controlled by a single recessive nucleic gene. Using 209 recessive mutant individuals in the F2 population derived from the cross Pei＇ai 64S × Qiufeng M, a gene, tentatively named gra（t）, was located between the SSR markers of RM475 and RM2-22 on the long arm of chromosome 2. The genetic distance were 17.3 cM and 2.9 cM respectively.

Marker_Free: a Novel Tendency of Transgenic Plants

Marker free is a rapidly developed strategy that offers a new approach for the elimination of public concerns caused by the selectable marker genes conferring antibiotic or herbicide resistance and so on. Furthermore, marker_free transgenic plants (MFTPs) have a number of special advantages, such as decreasing the concerns about safety of selectable marker and stacking transgenes progressively into transgenic plants, which significantly owns potential application value. Major approaches developed recently for obtaining MFTPs were reviewed in this paper.

Genetic Transformation of Nannochloropsis oculata with a Bacterial Phleomycin Resistance Gene as Dominant Selective Marker

The gene ble from Streptoalloteichus hindustanus is widely used as a selective antibiotic marker. It can control the phleomycin resistance, and significantly increase the tolerance of hosts to zeocin. The unicellular marine microalga Nannochloropsis oculata is extremely sensitive to zeocin. We selected ble as the selective marker for the genetic transformation of N. oculata. After the algal cells at a density of 2× 10^7 cells mL-1 was digested with 4% hemicellulase and 2% driselase for 1 h, the protoplasts accounted for 90% of the total. The ble was placed at the downstream of promoter HSP70A-RUBS2 isolated from Chlamydomonas reinhardtii, yielding a recombinant expression construct pMS 188. The construct was transferred into the protoplasts through electroporation （1 kV, 15 μS）. The transformed protoplasts were cultured in fresh f/2 liquid medium, and selected on solid f/2 medium supplemented with 500 ng mL-t zeoein. The PCR result proved that ble existed in the transformants. Three transformants had been cultured for at least 5 generations without losing ble. Southern blotting analysis showed that the ble has been integrated into the genome of N. oculata. The ble will serve as a new dominant selective marker in genetic engineering N. oculata.

Polymorphisms in the Myostatin-1 gene and their association with growth traits in Ancherythroculter nigrocauda

Myostatin （MSTN） is a member of the transforming growth factor-β gene superfamily that negatively regulates skeletal muscle development and growth. In the present study, partial genomic fragments of Myostatin-1 （MSTN-1） in two commercial hatchery populations of Ancherythroculter nigrocauda, an economically important freshwater fish, were screened for single nucleotide polymorphisms （SNPs） and then genotyped by direct sequencing of PCR products. Five SNPs were identified in intron 1 and exon 2, including a non-synonymous mutation causing an amino acid change （Val to Ile） at position 180. Association analyses based on 300 individuals revealed that the g. 1129T〉C SNP locus was significantly associated with total length （TL）, body length （BL）, body height （BH） and body weight （BW） in 6- and 18-month-old populations, while the g. 1289G〉A locus was significantly associated with BH and BW in the 6-month-old population. Haplotype analyses revealed that fish with the genotype combinations TC/TC or TC/GA showed better growth performance. Our results suggest that g.l129T〉C and g.1289G〉A have positive effects on growth traits and may be candidate gene markers for marker-assisted selection in A. nigrocauda.

SMAD7 and SERPINE1 as novel dynamic network biomarkers detect and regulate the tipping point of TGF-beta induced EMT

Epithelial–mesenchymal transition(EMT) is a complex nonlinear biological process that plays essential roles in fundamental biological processes such as embryogenesis, wounding healing, tissue regeneration,and cancer metastasis. A hallmark of EMT is the switch-like behavior during state transition, which is characteristic of phase transitions. Hence, detecting the tipping point just before mesenchymal state transition is critical for understanding molecular mechanism of EMT. Through dynamic network biomarkers(DNB) model, a DNB group with 37 genes was identified which can provide the early-warning signals of EMT. Particularly, we found that two DNB genes, i.e., SMAD7 and SERPINE1 promoted EMT by switching their regulatory network which was further validated by biological experiments. Survival analyses revealed that SMAD7 and SERPINE1 as DNB genes further acted as prognostic biomarkers for lung adenocarcinoma.