Effects of five cryoprotectants on proliferation and differentiation‐related gene expression of frozen‐thawed bovine calf testicular tissue

The cryopreservation of testicular tissue is a potential method for preserving male fertility. However, the effect of cryopreservation on bovine calf testicular tissue is scarce. This study investigated the effect of different cryoprotectants on bovine calf testicular tissue at the molecular level. Testicular tissue from ten immature bovine calves (6 months) was collected after slaughter and cryopreserved in an extender containing different concentrations of the following five cryopreservation solutions (CP): bovine serum albumin (BSA) with 5% dimethyl sulfoxide (DMSO), trehalose with 5% DMSO, DMSO and glycerol and ethylene glycol (EG). After 7‐day cryopreservation, the expression levels of three spermatogonial stem cell (SSC)‐related genes, octamer‐4 (OCT4), KIT ligand (MGF/SCF) and kit oncogene (C‐KIT), were investigated by quantitative PCR (qPCR). The cell viability was highest for the tissues preserved with 30 mg/ml BSA (77.82% ± 1.22) and 40 mg/ml trehalose (74.23% ± 1.16) compared with other groups (p < 0.05), and the level of expression of the three genes was highest with 30 mg/ml BSA (p < 0.05). Compared with other CPs, the 30 mg/ml BSA and 40 mg/ml trehalose have the better cryopreserve protection. The 30 mg/ml BSA is the most viable media for the cryopreservation of testicular tissue from cattle.     

青花菜锌指蛋白基因BoCCCH1的克隆和表达分析

CCCH锌指蛋白广泛存在于真核生物中,在植物的生长、发育和逆境胁迫响应中起着重要作用。为获知青花菜CCCH转录因子基因的序列特征和表达特性,从青花菜叶片中克隆到1个CCCH基因,命名为Bo CCCH1,同时,利用RT-PCR方法研究了其在不同器官及霜霉菌侵染叶片中的表达模式。序列分析结果表明,Bo CCCH1的基因组全长为1 568 bp,包含1个长度为599 bp的内含子,2个长度分别为627 bp、342 bp的外显子,编码322个氨基酸,蛋白质的分子量与等电点分别为35 296.42 Da和8.47;Bo CCCH1有2个锌指结构,类型均为C-X8-C-X5-C-X3-H。系统发育分析结果表明,Bo CCCH1与其它植物的21条同源序列在进化树上分为6组,来自十字花科的CCCH与Bo CCCH1处于同一分支。基因表达结果表明,Bo CCCH1在叶、花茎、嫩角果、花蕾和花中表达,其中花茎和嫩角果的表达量最高,但在根中未检测到表达;在霜霉菌侵染下,叶片Bo CCCH1的表达量升高,到24 h和36 h时达到最大,推测Bo CCCH1与霜霉病抗性相关。本研究为进一步探明Bo CCCH1在霜霉病抗性反应中的功能奠定了基础。     

Improvements of TKC Technology Accelerate Isolation of Transgene-Free CRISPR/Cas9-Edited Rice Plants

Elimination of the CRISPR/Cas9 constructs in edited plants is a prerequisite for assessing genetic stability, conducting phenotypic characterization, and applying for commercialization of the plants. However, removal of the CRISPR/Cas9 transgenes by genetic segregation and by backcross is laborious and time consuming. We previously reported the development of the transgene killer CRISPR (TKC) technology that uses a pair of suicide genes to trigger self-elimination of the transgenes without compromising gene editing efficiency. The TKC technology enables isolation of transgene-free CRISPR-edited plants within a single generation, greatly accelerating crop improvements. Here, we presented two new TKC vectors that show great efficiency in both editing the target gene and in undergoing self-elimination of the transgenes. The new vectors replaced the CaMV35S promoter used in our previous TKC vector with two rice promoters to drive one of the suicide genes, providing advantages over our previous TKC vector under certain conditions. The vectors reported here offered more options and flexibility to conduct gene editing experiments in rice.     

Identification of Differentially Expressed Genes and Lipid Metabolism Signaling Pathways between Muscle and Fat Tissues in Broiler Chickens

In this study, signaling pathways and key differentially expressed genes (DEGs) involved in lipid metabolism in muscle and fat tissues were investigated. Muscle and abdominal fat tissues were obtained from 35-day-old female broilers for RNA sequencing. DEGs between muscle and fat tissues were identified. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses of DEGs were performed. A total of 6130 DEGs were identified to be significantly enriched in 365 GO terms, most of which were involved in biological processes, cellular components, and molecular functions in muscle and fat tissues. Three important lipid signaling pathways (pyruvate metabolism, the insulin signaling pathway, and the adipocytokine signaling pathway) were identified among the fat and muscle tissues of broilers. The key common DEGs in these pathways included phosphoenolpyruvate carboxykinase 2 (PCK2), acetyl-CoA carboxylase 1 alpha and beta (ACACA and ACACB), and the mitogen-activated protein kinase (AMPK) gene family. Hence, our findings revealed the pathways and key genes and gene families involved in the regulation of fat deposition in the muscle and fat tissues of broilers.     

UCP1基因变异与绵羊生长及胴体性状的关联分析

试验旨在探讨解耦联蛋白1（UCP1）基因核苷酸变异对绵羊生长性状的性别差异和胴体性状的影响，以期能够筛选出可以提升绵羊生长及胴体性状的核苷酸变异，为提高绵羊相关重要经济性状的分子遗传标记提供材料。以9个绵羊品种为研究对象，用PCR-SSCP方法检测不同性别绵羊中UCP1基因内含子5区和外显子6区变异。利用Minitab 16.0软件中一般线性模型分析内含子5区等位基因与不同性别绵羊生长性状、公羔胴体性状的关联性。结果显示，绵羊UCP1基因内含子5区和外显子6区共检测到8个核苷酸变异，其中位点c.910 G/A突变导致p.Ala304Thr氨基酸变异。生长性状关联分析结果表明，内含子5区等位基因对绵羊生长性状的影响存在性别特异性，母羔中携带等位基因A₁的群体较缺失群体具有较低的初生重（P<0.05），公羔中携带等位基因C₁的群体较缺失群体具有较高的断尾重（P<0.05），未发现其他等位基因与羔羊的生长性状存在性别特异性。胴体性状关联分析结果表明，携带等位基因A₁的群体具有较低后腿瘦肉量、腰部瘦肉量和较高的肩部瘦肉比例（P<0.05），携带等位基因C₁的群体具有较低的后腿瘦肉比例（P<0.05），其他胴体性状均没有发现与等位基因存在显著相关，基因型分析结果与等位基因分析结果一致。结果提示，UCP1基因对绵羊的生长性状影响具有性别特异性，且携带等位基因A₁的公羔群体具有较低的胴体生产性状，为提高公羔胴体生产性能提供了理论依据。     

Cloning of Buffalo BMP1 Gene and its Expression Pattern Investigation in Different Tissues

Buffalo BMP1 gene was cloned in the present study, the BMP1 sequence was systemically analysed by bioinformatics techniques, and the expression level of BMP1 gene in different tissues were also assayed with Real-time fluorescence quantitative PCR (QRT-PCR).The results showed that with RT-PCR a 3 195 bp buffalo BMP1 gene was cloned and sequenced, including the whole ORF of 2 967 bp (coding 988 amino acid).The sequence multialigned results showed that buffalo BMP1 gene shared 99%, 96%, 96%, 96% and 95% of similar amino acid sequence with Bos taurus, Sus scrofa, Equus, Homa sapiens and Mus musclus, respectively.It was predicted that buffalo BMP1 protein contained a signal peptide domain, a preregion, a metalloproteinases domain, five complement-Uegf-BMP-1 domain (CUB domain) and two epitheloid growth factor-like domain (EGF-like domain).In addition, we also analyzed the expression level in different tissues through QRT-PCR, the results showed that BMP1 gene mRNA existed in all nine tissues with the most abundant expression in heart, followed by testis, ovary, genital ridge, while with lower amount of bone and other tissues, the minimal expression in liver was observed.