Journal of Genetics and Genomics

Genetic Transformation of Aloe barbadensis Miller by Agrobacterium tumefaciens

Congfen He, Jiaxing Zhang, Jie Chen, Xingguo Ye, Lipu Du, Yinmao Dong, Hua Zhao

2007, 34(12): 1053-1060. doi: 10.1016/S1673-8527(07)60120-9

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Abstract:
Despite the importance of aloe in cosmetic and pharmaceutical industries, improvement of aloe (Aloe barbadensis Miller) by genetic engineering was seldom reported previously. In this study, regeneration and transformation conditions, including explant selection and surface sterilization, use of different Agrobacterium strains, and co-culture processing, are optimized. The use of 20.0% sodium hypochloride (25 min) for sterilization was less detrimental to the health of explant than 0.1% mercuric chloride (10 min). Regeneration frequency from stems was much higher than that from leaves or sheaths. Explants were infected by Agrobacterium (30 min) in liquid co-cultural medium, and this was followed by three days co-culture on sterile filter papers with light for 10 h per day at 24°C. Histochemical data demonstrated that the transient expression of GUS gene in the stem explants of aloe infected with Agrobacterium strains EHA105 and C58C1 was 80.0% and 30.0%, respectively, suggesting the higher sensitivity of the explants to EHA105 than to C58C1. Infected tissues were selected using G418 (10.0–25.0 mg/L) to generate transformants. Sixty-seven G418 resistant plantlets were generated from the infected explants. Southern blotting, PCR, and ELISA analyses indicated that the alien gene were successfully transferred into aloe and was expressed in the transgenic plants. This newly established transformation system could be used for the genetic improvement of aloe.

Population Genetic Diversity in Chinese Pomegranate (Punica granatum L.) Cultivars Revealed by Fluorescent-AFLP Markers

Zhaohe Yuan, Yanlei Yin, Jianlu Qu, Liqin Zhu, Yun Li

2007, 34(12): 1061-1071. doi: 10.1016/S1673-8527(07)60121-0

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Eighty-five pomegranate (Punica granatum L.) cultivars from six geographical populations located at Shandong, Anhui, Shaanxi, Henan, Yunnan, and Xinjiang Provinces were studied for its population genetic diversity by means of fluorescent-AFLP markers. The results indicated that 135–185 polymorphic loci were amplified by eight pairs of primers at species level. An average of 158.25 polymorphic loci was amplified for each primer combination. The polymorphism percentage ranged from 62.5% to 86.11%, and the average polymorphism percentage was 73.26%. This indicated that there was plentiful genetic diversity in pomegranate cultivars. The genetic diversity at the species level was higher than that at the population level. The order of the genetic diversity was Henan population > Xinjiang population > Shaanxi population > Anhui population > Shandong population > Yunnan population. Variance analysis showed that there was significant difference between populations in genetic diversity. The genetic differentiation coefficient between populations ( G_ST) was 0.2018, which indicated that gene differentiation was mainly within the population, and between populations, it accounted for 20.18% of the total variation. Gene flow (Nm) between the populations measured was 1.9027 based on the genetic differentiation coefficient between populations, indicating that there was mild gene flow between populations. The UPGMA cluster analysis showed that most accessions from the same population were clustered together, but there was partly gene exchange. All genetic parameters indicated that there was plentiful genetic diversity in pomegranate cultivars in China, of which Henan population was significantly higher than the other populations, and it had wide application foreground in pomegranate breeding in China.

Genetic Relationships Among Four Minorities in Guangxi Revealed by Analysis of 15 STRs

Qiongying Deng, Lin Xu, Jichun Gong, Lining Zhou, Songfeng Li, Xiangfa Deng, Guorong Luo, Xiaoxun Xie

2007, 34(12): 1072-1079. doi: 10.1016/S1673-8527(07)60122-2

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Abstract:
The aim of this study is to investigate the genetic diversity in 15 STRs (short tandem repeats) loci of four minorities in Guangxi Province and to probe into the genetic variation and relationships among these ethnic groups. Allele frequencies of 15 STR loci were collected from 766 unrelated Mulao, Maonan, Miao, and Yao ethnic individuals by PCR-STR and sequencing, and their allele-frequency distribution were compared with each other. The genetic parameters and genetic distances were calculated, and the phylogenetic tree was constructed. Based on the results from this study, 135, 134, 148, and 145 alleles and 424, 432, 445, and 436 genotypes for 15 STR loci were observed in the Mulao, Maonan, Miao, and Yao minorities, respectively. The average heterozygosity of all ethnic groups analyzed was above 0.7; the cumulative power of discrimination (DP), the probabilities of paternity exclusion (EPP), and the polymorphic information content (PIC) were greater than 0.99999. Comparison of the allele-frequency distribution indicated that there were significant differences at most loci between Maonan vs. Miao, Yao vs. other groups, but no distinct differences between Mulao vs. Maonan, and Mulao vs. Miao minorities. The NJ tree based on the genetic distance showed that the four minorities were separated into two groups. Mulao and Maonan were clustered into one group, whereas Miao and Yao into the other. Our results revealed that 15 STR loci of the four minorities possessed high genetic diversities. Therefore, the combination of these 15 STRs is a powerful tool for forensic individual identification and paternity investigation, as well as anthropologic and genetic researches. The genetic variation and relationships among the 4 populations revealed by 15 STRs are basically consistent with their linguistic culture and ethical history.

Prediction of Subcellular Localization of Eukaryotic Proteins Using Position-Specific Profiles and Neural Network with Weighted Inputs

Lingyun Zou, Zhengzhi Wang, Jiaomin Huang

2007, 34(12): 1080-1087. doi: 10.1016/S1673-8527(07)60123-4

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Abstract:
Subcellular location is one of the key biological characteristics of proteins. Position-specific profiles (PSP) have been introduced as important characteristics of proteins in this article. In this study, to obtain position-specific profiles, the Position Specific Iterative-Basic Local Alignment Search Tool (PSI-BLAST) has been used to search for protein sequences in a database. Position-specific scoring matrices are extracted from the profiles as one class of characteristics. Four-part amino acid compositions and 1st–7th order dipeptide compositions have also been calculated as the other two classes of characteristics. Therefore, twelve characteristic vectors are extracted from each of the protein sequences. Next, the characteristic vectors are weighed by a simple weighing function and inputted into a BP neural network predictor named PSP-Weighted Neural Network (PSP-WNN). The Levenberg-Marquardt algorithm is employed to adjust the weight matrices and thresholds during the network training instead of the error back propagation algorithm. With a jackknife test on the RH2427 dataset, PSP-WNN has achieved a higher overall prediction accuracy of 88.4% rather than the prediction results by the general BP neural network, Markov model, and fuzzy k-nearest neighbors algorithm on this dataset. In addition, the prediction performance of PSP-WNN has been evaluated with a five-fold cross validation test on the PK7579 dataset and the prediction results have been consistently better than those of the previous method on the basis of several support vector machines, using compositions of both amino acids and amino acid pairs. These results indicate that PSP-WNN is a powerful tool for subcellular localization prediction. At the end of the article, influences on prediction accuracy using different weighting proportions among three characteristic vector categories have been discussed. An appropriate proportion is considered by increasing the prediction accuracy.

cDNA Cloning, Bioinformatic and Tissue-specific Expression Analysis of Porcine JARID1C Gene

Lu Yi, Zhenhua Hao, Tongtong Yang, Shaobing Wang, Baosong Xing, Yinxue Xu

2007, 34(12): 1088-1096. doi: 10.1016/S1673-8527(07)60124-6

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Jumonji, AT-rich interactive domain 1C (JARID1C) protein belongs to the highly conserved ARID protein family, which is involved in chromatin remodeling and transcriptional regulation during cell growth, differentiation, and development. In humans, this gene plays a vital role in normal brain development and function. Using an in silico approach in combination with 5′ rapid amplification of cDNA ends (5′ RACE), the full-length cDNA of JARID1C (GenBank accession No. EF139241) from porcine ovary, which contains 5,908 bp nucleotides, with an open reading frame (ORF) of 4,548 bp, has been cloned. The putative porcine JARID1C protein, which is located in the nucleus, encodes 1,516 amino acids with a molecular weight of 170 kDa and a pI of 5.44. Bioinformatic prediction indicates that the protein contains several conserved domains: a JmjN domain, an ARID domain, a JmjC domain, a C5HC2 zinc finger domain, and a PHD zinc finger domain. Similarity comparisons for nucleic and amino acid sequences reveal that the porcine JARID1C protein shares a high identity with its dog, mouse, rat, and human counterparts. The phylogenetic tree of the JARID1 subfamily proteins has been constructed to reveal the evolutionary relationship of various species. Real-time PCR analysis shows that theJARID1C gene is expressed in various tissues, but at different levels. The expression levels of this gene are higher in the brain and gonad than in other tissues, suggesting that the JARID1C protein plays a role in porcine brain and gonad functions.

Analysis on the Origin and Phylogenetic Status of Tong Sheep Using 12 Blood Protein and Nonprotein Markers

Wei Sun, Hong Chang, Zhangping Yang, Rongqing Geng, Kenji Tsunoda, Zhanjun Ren, Hongyu Chen, Musa H. Hussein

2007, 34(12): 1097-1105. doi: 10.1016/S1673-8527(07)60125-8

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This study is based on the Tong sheep obtained by the random sampling method of typical colonies in the central area of Baishui County in Shaanxi Province, China. An investigation was undertaken to clarify the gene constitution of blood protein and nonprotein types of Tong sheep. Twelve genetic markers were examined by starch-gel electrophoresis and cellulose acetate electrophoresis. Polymorphism in Tong sheep was found at the following 10 loci, transferrin (Tf), alkaline phosphatase (Alp), leucine aminopeptidase (Lap), arylesterase (Ary-Es), hemoglobin-β (Hb-β), X-protein (X-p), carbonic anhydrase (CA), catalase (Cat), malate dehydrogenase (MDH), and lysine (Ly), whereas, albumin (Al) and postalbumin (Po) loci were monomorphic. Genetic approach degree method and phylogenetic relationship clustering method were used to judge the origin and phylogenetic status of Tong sheep. Results from both methods maintained that Tong sheep belonged to the “Mongolia group”, and Mongolia sheep was the origin of Tong sheep. This was also supported by the history of Tong sheep breeding. Compared to the phylogenetic relationship clustering method, the genetic approach degree method was more reliable for the extraction from East and South of Central Asia, and was more effective in reflecting the breeding course of Tong sheep.

Effects of Downregulation of Inhibin α Gene Expression on Apoptosis and Proliferation of Goose Granulosa Cells

Fengjian Chen, Xunping Jiang, Xiuping Chen, Guiqiong Liu, Jiatong Ding

2007, 34(12): 1106-1113. doi: 10.1016/S1673-8527(07)60126-X

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Inhibin α is one of the candidate genes that control the ovulation in poultry. To study the genetic effects of inhibin α on apoptosis and proliferation of goose granulosa cells cultured in vitro, two RNA interference (RNAi) expression vectors, psiRNA-INHα1 and psiRNA-INHα2, were constructed to knock down inhibin α gene expression. After 48 h of transfection, the efficiency of these two RNAi expression vectors was examined by fluorescence microscopy. Meanwhile, inhibin protein expression levels, apoptosis indexes (AI) and proliferation indexes (PI) of granulosa cells were analyzed by flow cytometry. In addition, the supernatants were collected to assay the concentrations of estrogen (E₂) and progesterone (P) by radioimmunoassay. The results showed that the expression level of inhibin α in the RNAi group were decreased 30%–40% than those in the control groups (P < 0.05) and the apoptosis indexes and proliferation indexes in the RNAi groups were significantly higher than those in the control groups ( P < 0.05). However, the E ₂ concentrations in the RNAi groups were lower than those in the control groups (P < 0.05). These results indicate that inhibin α has antagonistic effect on granulosa cell apoptosis.

Genetic Variation of Wild and Hatchery Populations of the Pacific Oyster Crassostrea gigas Assessed by Microsatellite Markers

Hong Yu, Qi Li

2007, 34(12): 1114-1122. doi: 10.1016/S1673-8527(07)60127-1

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Microsatellite DNA technique was used to detect the genetic variation between five hatchery populations of the Pacific oyster from China and two wild populations from Japan. Seven microsatellite loci screened in this study showed high polymorphism in both hatchery and wild populations, as observed in an average number of allele per locus (19.1–29.9) and average expected heterozygosity (0.916–0.958). No significant difference in average allelic richness or expected heterozygosity was observed between Chinese hatchery populations and Japanese wild populations. Pairwise values and heterogeneity tests of allele frequencies showed significant genetic differentiation between all populations. According to the neighbor-joining tree constructed on the basis of theD_C distance, the seven populations fell into three groups showing a clear division between hatchery and wild populations, and between the northern and southern hatchery populations. Assignment tests correctly assigned high percentages (97%–100%) of individuals to their original populations and demonstrated the feasibility of microsatellite analysis for discrimination between populations. The information obtained in this study is useful for designing suitable management guidelines and selective breeding programs for the Pacific oyster in China.

Mapping of a Major Stripe Rust Resistance Gene in Chinese Native Wheat Variety Chike Using Microsatellite Markers

Fanghui Liu, Yongchun Niu, Hui Deng, Genjia Tan

2007, 34(12): 1123-1130. doi: 10.1016/S1673-8527(07)60128-3

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Chike (accession number Su1900), a Chinese native wheat (Triticum aestivum L.) variety, is resistant to the currently prevailing physiological races of Puccinia striiformis Westend. f. sp. tritici in China. Genetic analysis indicated that resistance to the physiological race CY32 of the pathogen in the variety was controlled by one dominant gene. In this study, BSA (bulked segregant analysis) methods and SSRs (simple sequence repeats) marker polymorphic analysis are used to map the gene. The resistant and susceptible DNA bulks were prepared from the segregating F₂ population of the cross between Taichung 29, a susceptible variety as maternal parent, and Chike as paternal parent. Over 400 SSR primers were screened, and five SSR markers Xwmc44, Xgwm259, Xwmc367, Xcfa2292, and Xbarc80 on the chromosome arm 1BL were found to be polymorphic between the resistant and the susceptible DNA bulks as well as their parents. Genetic linkage was tested on segregating F₂ population with 200 plants, including 140 resistant and 60 susceptible plants. All the five SSR markers were linked to the stripe rust resistance gene in Chike. The genetic distances for the markers Xwmc44, Xgwm259, Xwmc367, Xcfa2292, and Xbarc80 to the target gene were 8.3 cM, 9.1 cM, 17.2 cM, 20.6 cM, and 31.6 cM, respectively. Analysis using 21 nulli-tetrasomic Chinese Spring lines further confirmed that all the five markers were located on chromosome 1B. On the basis of the above results, it is reasonable to assume that the major stripe rust resistance gene YrChk in Chike was located on the chromosome arm 1BL, and its comparison with the other stripe rust resistance genes located on 1B suggested that YrChk may be a novel gene that provides the resistance against stripe rust in Chike. Exploration and utilization of resources of disease resistance genes in native wheat varieties will be helpful both to diversify the resistance genes and to amend the situation of resistance gene simplification in the commercial wheat cultivars in China.

Disrupted ompC Causes Osmosis Sensitivity of Escherichia coli in Alkaline Medium

Yuqi Wang, Lingling Wang, Yirong Sun, Yicai Chen, Lei Zhu, Lixia Guo, Biao Luo, Haihong Wang

2007, 34(12): 1131-1138. doi: 10.1016/S1673-8527(07)60129-5

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The Escherichia coli strain DH42 is sensitive to high osmolarity in an alkaline medium. Using mini-Tn5 mutagenesis, construction of mutant strains by homologous recombination and subcloning of DNA fragment techniques, gene ompC was identified as the key factor that, once disrupted, caused osmosis-sensitivity ofE. coli strain DH42 grown in an alkaline medium. Through P1 transduction, a mutant strain, D9 (W3110 ompC:kan), was constructed and growth comparison was performed between DH42 and D9 under different pHs and salt concentrations. The result showed that ompC was necessarily required for hyperosmotic adaptation of E. coli in the alkaline medium.

2007 Vol. 34, No. 12