Sequence-length variation of mtDNA HVS-I C-stretch in Chinese ethnic groups

Sequence-length variation of mtDNA HVS-I C-stretch in Chinese ethnic groups 1 3 4 first-author 1 1 2 1 3 4 1 2 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:chenteng@mail.xjtu.edu.cn †E-mail:chenteng@mail.xjtu.edu.cn chenteng@mail.xjtu.edu.cn Department of Forensic Medicine, School of Medicine, Xi’an Jiaotong University, Xi’an 710061, China Department of Forensic Medicine, School of Medicine, Xi’an Jiaotong University, Xi’an 710061, China Ministry of Education Key Laboratory of Environment and Genes Related to Diseases, Xi’an Jiaotong University, Xi’an 710061, China Ministry of Education Key Laboratory of Environment and Genes Related to Diseases, Xi’an Jiaotong University, Xi’an 710061, China Beijing Genomics Institute, Chinese Academy of Sciences, Beijing 100029, China Beijing Genomics Institute, Chinese Academy of Sciences, Beijing 100029, China Medical College of Georgia, 1459 Laney Walker Blvd, Augusta, Georgia 30912, USA Medical College of Georgia, 1459 Laney Walker Blvd, Augusta, Georgia 30912, USA The purpose of this study was to investigate mitochondrial DNA (mtDNA) hypervariable segment-I (HVS-I) C-stretch variations and explore the significance of these variations in forensic and population genetics studies. The C-stretch sequence variation was studied in 919 unrelated individuals from 8 Chinese ethnic groups using both direct and clone sequencing approaches. Thirty eight C-stretch haplotypes were identified, and some novel and population specific haplotypes were also detected. The C-stretch genetic diversity ( GD ) values were relatively high, and probability ( P ) values were low. Additionally, C-stretch length heteroplasmy was observed in approximately 9% of individuals studied. There was a significant correlation ( r =−0.961, P < 0.01) between the expansion of the cytosine sequence length in the C-stretch of HVS-I and a reduction in the number of upstream adenines. These results indicate that the C-stretch could be a useful genetic maker in forensic identification of Chinese populations. The results from the Fst and dA genetic distance matrix, neighbor-joining tree, and principal component map also suggest that C-stretch could be used as a reliable genetic marker in population genetics. Mitochondrial DNA (mtDNA) Clone sequencing Length heteroplasmy Population genetics INTRODUCTION 1 Mitochondrial DNA (mtDNA) is well suited for forensic genetics, molecular anthropology, and medicine studies because of its stability, lack of recombination, rapid evolutionary changes, and high population specific polymorphisms (Anderson et al., 1981 1981 ; Brown et al., 1982 1982 ; Bowling et al., 1993 1993 ; Allen et al., 1998 1998 ; Macaulay et al., 1999 1999 ; Ingman et al., 2000 2000 ). The mtDNA sequence evolves much faster than nuclear DNA (Brown et al., 1982 1982 ). A great number of mtDNA variations have been defined, including transition, transversion, deletion, inversion, insertion, and complex rearrangement (Brandon et al., 2005 2005 ). MtDNA hypervariable segment I (HVS-I) contains a C-continuous tract termed the C-stretch, which is associated with sequence-length variations (Lee HY et al., 2004 2004 ; Lutz-Bonengel et al., 2004 2004 ) located in 16 180~16 193 nt. Due to slipping of the DNA polymerase during replication, the C-stretch evolves much faster than other regions of mtDNA, and variations in this region have been demonstrated widely among unrelated individuals (Malik et al., 2002a 2002a ; Lee HY et al., 2004 2004 ; Lutz-Bonengel et al., 2004 2004 ). Furthermore, the mtDNA control region, especially the C-stretch, might be involved in the development of human diseases such as cancer (Tan et al., 2002 2002 ; Lee HC et al., 2004 2004 ; Meierhofer et al., 2004 2004 ; Montanini et al., 2005 2005 ; Sangkhathat et al., 2005 2005 ). C-stretch length heteroplasmy within both HVS-I and HVS-II has been observed, and the rates of heteroplasmic length variation revealed significant differences among distinct populations (Salas et al., 2001 2001 ; Chen et al., 2002 2002 ; Imaizumi et al., 2002 2002 ; Mabuchi et al., 2007 2007 ). An individual can exhibit two or more different C-stretch lengths in different tissues (Kirches et al., 2001 2001 ; Lee et al., 2006 2006 ; Lutz-Bonengel et al., 2008 2008 ). Furthermore, even in the same tissue, especially in hairs, C-stretch length heteroplasmy was demonstrated in different hair shafts (Pfeiffer et al., 2004 2004 ; Lee et al., 2006 2006 ) and even in different parts of the same hair (Salas et al., 2001 2001 ). As the ‘out-of-phase’ nucleotide pattern, C-stretch is not easily detected when compared to other regions of mtDNA, and C-stretch length heteroplasmy increases the difficulties of DNA sequencing (Lee HY et al., 2004 2004 ; Lutz-Bonengel et al., 2004 2004 ; Bini and Pappalardo, 2005 2005 ). C-stretch is located in the middle of mtDNA HVS-I, and failure to interpret these sequence variations may hinder the application of the mtDNA control region to forensic and population genetics (Malik et al., 2002a 2002a ; Lee HY et al., 2004 2004 ). Therefore, the C-stretch might be highly significant in forensic identification and population genetic studies. In the present study, we investigated the mtDNA HVS-I C-stretch sequence-length variation in 919 unrelated individuals from 8 Chinese ethnic groups by using direct and clone sequencing approaches and explored the significance of these findings to forensic, population genetics, and medicine studies. MATERIALS AND METHODS 1 Sample collection 2 A total of 919 unrelated healthy individuals from 8 different Chinese ethnic groups were analyzed for C-stretch haplotypes in the mtDNA HVS-I region. Northern Han ( N =188) samples were obtained from Xining, Xi’an and Beijing. Southern Han ( N =117) samples were from Kunming, Wuhan and Zhanjiang. Tu ( N =146), Tibetan ( N =120) and Salar ( N =100) were all collected from Qinghai Province. Oroqen ( N =50) samples were collected from Inner Mongolia. Yugur ( N =100) and Bonan ( N =98) were collected from Gansu Province. The locations and the language families of the studied ethnic groups are shown in Table 1 1 and Fig. 1 1 (Du and Yip, 1993 1993 ; Ma, 1994 1994 ). The geographic origin, nationality, and maternal pedigree (unrelated at least through three generations) of each individual were ascertained at first. Informed consent was obtained before sampling. The whole blood samples were obtained by venipuncture and put into ethylenediaminetetraacetic acid (EDTA) tubes. The language family is from Du and Yip ( 1993 1993 ) and Ma ( 1994 1994 ) The total population size of each population is based on the fifth national Census (2000) Population Location Longitude Latitude Language a a Census size b b Sample size 3 1. Northern Han Xining, Qinghai 101.75° E 36.57° N 3 Chinese, Sino-Tibetan family 1 979 200 37 Xi’an, Shaanxi 108.95° E 34.27° N 7 410 000 109 Beijing 116.42° E 39.92° N 14 640 000 42 3 2. Southern Han Kunming, Yunnan 102.73° E 25.05° N 3 Chinese, Sino-Tibetan family 5 781 000 43 Wuhan, Hubei 114.32° E 30.52° N 8 312 600 42 Zhanjiang, Guangdong 110.30° E 21.20° N 6 072 900 32 3. Tu Huzhu Tu autonomous county, Qinghai 101.90° E 36.80° N Mongolian group, Altaic family 199 470 146 4. Tibetan Yushu Tibetan autonomous prefectures, Qinghai 96.97° E 33.03° N Tibetan branch, Tibetan-Burman group, Sino-Tibetan family 1 134 236 120 5. Oroqen Hulun Buir League, inner Mongolia 123.70° E 50.58° N Tungus branch, Manchu-Tungusic group, Altaic family 8 196 50 6. Salar Xunhua Salar autonomous county, Qinghai 102.40° E 35.80° N Tujue branch, Altaic family 95 815 100 7. Yugur Yugur autonomous county, Gansu 99.60° E 38.80° N Turkic branch, Altaic family 13 719 100 8. Bonan Jishishan autonomous county, Gansu 102.80° E 35.70° N Mongolian branch, Altaic family 16 505 98 Total 2 919 DNA extraction, amplification, and direct sequencing 2 Genomic DNA was extracted using the Chelex-100 method as previously described (Walsh et al., 1991 1991 ). Polymerase chain reaction (PCR) was performed in a 25-μl reaction volume using GeneAmp PCR System 9700 (PE Applied Biosystems, Foster City, CA, USA) with primers L15990 (5′-ACTCCACCATTAGCACC-3′) and H16503 (5′-CAGATGTCGGATACAGTTC-3′). Each reaction mixture contained 50 mmol/L KCl, 10 mmol/L Tris-HCl, 1.5 mmol/L MgCl 2 , 50 mmol/L of dNTP each, 0.25 mmol/L of each primer, and 1.25 U AmpliTaqGold DNA polymerase. The thermal cycling conditions were 10 min at 95 °C followed by 36 cycles of 30 s at 95 °C, 30 s at 60 °C, and 45 s at 72 °C, and a final extension of 10 min at 72 °C. PCR products were purified by Centrisep (PE Applied Biosystems) and sequenced using BigDye® Terminator Cycle Sequencing Ready Reaction Kit (PE Applied Biosystems) according to the manufacturer’s manual. Then, the products were purified with DyeEx-columns (Qiagen, Hilden, Germany) and dried on a heat block. The dried specimens were dissolved in HiDi formamide and analyzed using ABI 3730 capillary electrophoresis (PE Applied Biosystems). Sequencing of both strands was performed to reduce ambiguities in sequence determination as recommended by the DNA Commission of the International Society for Forensic Genetics (Carracedo et al., 2000 2000 ). Cloning and sequencing 2 For samples that provided ambiguous results by direct sequencing, a clone sequencing approach was used to define the exact sequence of each sample. Firstly, the ambiguous samples were correctly selected and then amplicons were purified by 6% (w/v) denaturing polyacrylamide gel electrophoresis (Acr: Bis=19:1, urea as denaturant). PCR products (530 bp) were cloned into pGem-T Easy vector system (Promega, Madison, WI, USA) and more than 20 clones were selected for each sample. Plasmid DNA was extracted from bacterial cells using QIAprep Spin Miniprep Kit (Qiagen). Purified plasmids were sequenced by the above mentioned sequencing method. To decrease the artificial generation of sequencing errors by Taq DNA polymerase during PCR, Pfu DNA polymerase (Promega, Madison, WI, USA) was used for amplification of samples with length heteroplasmy, and two independently generated PCR products from each sample were ligated into pGem-T Easy vector for cloning and randomly selected clones were sequenced in two directions by ABI 3730 capillary electrophoresis. For samples with greater C number ( n > 13), junction primers, which bind to a part of the C-stretch and the first 2~4 bases downstream of the C-stretch region, were used in the experiment to facilitate sequencing. Data analysis 2 The edited data were aligned with the revised Cambridge Reference Sequence (rCRS) (Andrews et al., 1999 1999 ) using DNAStar software ver. 5.0. Haplotypes and gene diversities were estimated according to Nei ( 1987 1987 ). The population diversity indices were estimated by Arlequin software ver. 2000 (Schneider et al., 2000 2000 ) and Dispan computer program (Ota, 1993 1993 ). The probability ( P ) of two randomly selected individuals from a population having identical haplotype types was P =∑ f 2 and gene diversity ( GD ) was calculated based on the following formula: GD = n (1−∑ f 2 )/( n −1) (Nei, 1996 1996 ), in which n stands for the sample number and f is the frequency of each C-stretch haplotype (Stoneking et al., 1991 1991 ). A comparative analysis between our haplotype data and the previously published data (Watson et al., 1996 1996 ) was carried out by using Arlequin software ver. 2000 (Schneider et al., 2000 2000 ) and Dispan computer program (Ota, 1993 1993 ). The Africa data were obtained from 241 individuals from 9 ethnic groups at hospitals and rural medical clinics in Kenya, Nigeria, and Niger in Africa (Watson et al., 1996 1996 ). According to the Fst genetic distances, the phylogenetic tree was constructed based on Neighbor Joining (NJ) method using the molecular evolutionary genetics analysis (MEGA) software ver. 4.0 (Tamura et al., 2007 2007 ) and Dispan computer program (Ota, 1993 1993 ). The principal component analysis (PCA) of the studied populations and the correlation study were performed by using SPSS 11.0 software. RESULTS 1 C-stretch variation of HVS-I 2 MtDNA sequences from all the subjects were successfully obtained using both direct and clone sequencing approaches. In the HVS-I region, we observed 38 C-stretch haplotypes, and the most common one, which was identical with the Cambridge Reference Sequence, was AAAACCCCCTCCCC and was found in all Chinese ethnic groups (Table A1 A1 ). Novel haplotypes and population specific haplotypes were also detected. The C-stretch haplotypes (AAAACCCCCACCCCC and AAAAGCCCCTCCCC) in HVS-I region, which contained a mutation of T to A transversion at position 16 189 and a mutation of C to G transversion at position 16 184, respectively, were observed for the first time. The haplotype (AAAGCCCCCTCCCC) was identified for the first time in Chinese ethnic groups (Fig. A1 A1 ); and AAAACCCCTCCCCC and AACCCCCCTCCCCCC were population specific haplotypes detected in Chinese Tibetan and Salar ethnic groups. Approximately 25.14% of all the samples were found to show T to C transition at position 16 189. The gene diversity values of HVS-I C-stretch haplotype in the studied ethnic groups were 0.3989, 0.7290, 0.6614, 0.4908, 0.5331, 0.6814, 0.5756 and 0.5803 across the various ethnic groups (Table A1 A1 ). This finding has been deposited into the GenBank database (accession numbers: EU920130 to EU920667). The novel mutations have been submitted to MITOMAP ( http://www.mitomap.org/ http://www.mitomap.org/ ), and the sequence numbers are from 20080730001 to 20080730003. Length heteroplasmy and correlation study of C-stretch 2 In the present study, C-stretch length heteroplasmy was detected in HVS-I region of approximately 9% of all the samples, which occurred exclusively in the samples with T to C transition at position 16 189. For the samples with C-stretch length heteroplasmy, we chose the average C-length of all clones as the representative of C-length for these samples (Table 2 2 and Fig. 2 2 ). For the samples with T to C transition at position 16 189 in HVS-I region, we analyzed the correlation between the expansion of cytosine length in the C-stretch and the reduction of adenines upstream to it, and the result shows a significant correlation ( r =−0.961, P < 0.01). Average C-length=(7×C7+8×C8+9×C9+10×C10+11×C11+12×C12)/20 Categorized into three characteristic types (Types I, II, and III, as shown in Fig. 2 2 ). Type I contains a predominant sequence type and no unreadable sequence. Type II shows a predominant sequence type and the unreadable sequence downstream. Type III contains two or more predominant sequence types and shows severely blurred sequences Cloning samples that were amplified and cloned again The values closed to 0.5 may be inconclusive results because two or more predominant types of C-length seem to be equally represent in direct sequencing Sample Predominant sequence type by direct sequencing C7 C8 C9 C10 C11 C12 Total clone Average C-length a a Sequence type b b  A   C7 19 1 0 0 0 0 20 7.1 I  B   C8 0 17 2 1 0 0 20 8.2 I  C   C9 0 1 16 3 0 0 20 9.1 I  D   C10 0 0 2 16 1 1 20 10.1 I  E   C7/C8 13 6 1 0 0 0 20 7.4 d d II  F   C9/C10 0 0 10 9 1 0 20 9.6 d d III  G c c   C8 0 20 0 0 0 0 20 8.0 I  H c c   C9 0 0 20 0 0 0 20 9.0 I Phylogenetic analysis and principal component analysis of populations 2 The Fst and dA genetic distance matrix of the eight Chinese populations and native African population is shown in Table 3 3 . The two different genetic distances showed a very close correlation ( r =0.853, P < 0.01). All pairwise Fst comparisons between African and eight Chinese populations were significant with values ranging between 0.1599 (African vs Southern Han) and 0.3105 (African vs Northern Han). There were no significant differences of Tu vs Oroqen ( Fst =0.0129, P > 0.05), Tu vs Bonan ( Fst =0.0089, P > 0.05), Oroqen vs Bonan ( Fst =0.0044, P > 0.05), or Salar vs Southern Han ( Fst =−0.0025, P > 0.05), which indicates that they are closely related populations. Fst values are shown below diagonal, and dA values above diagonal P > 0.05 Han (N) Han (S) Tu Tibetan Oroqen Salar Yugur Bonan African Han (N) 0.1266 0.1115 0.0929 0.1198 0.1128 0.1176 0.0678 0.3816 Han (S) 0.1015 0.1690 0.2587 0.1803 0.0739 0.1847 0.0869 0.4479 Tu 0.0515 0.0255 0.1236 0.1062 0.1444 0.2559 0.1128 0.4385 Tibetan 0.0192 0.0798 0.0191 0.1682 0.2216 0.2363 0.1524 0.4220 Oroqen 0.0613 0.0427 0.0129 * * 0.0510 0.1687 0.3334 0.0859 0.5478 Salar 0.0738 −0.0025 * * 0.0178 0.0569 0.0375 0.1821 0.0547 0.4566 Yugur 0.0373 0.0413 0.0412 0.0494 0.0685 0.0300 0.1740 0.3360 Bonan 0.0267 0.0268 0.0089 * * 0.0257 0.0044 * * 0.0138 0.0222 0.3766 African 0.3105 0.1599 0.1909 0.2609 0.2326 0.1810 0.2079 0.2190 Based on the Fst value (Table 3 3 ), an unrooted NJ tree (Fig. 3 3 ) of the eight Chinese populations and native African population was constructed. The eight Chinese populations showed a close affinity. The two Gansu populations (Yugur and Bonan) clustered together in the tree, the same as Tu and Salar (Qinghai populations). The genetic distances between Tibetan and the other Qinghai populations (Tu and Salar) were relatively far, which could be expected as the Tibetan population belong to a different language family (Sino-Tibetan family) (Table 1 1 ). Han (N) represents Northern Chinese Han ethnic group; Han (S) represents Southern Chinese Han ethnic group Fig. 4 4 displays the principal component (PC) analysis map of the nine populations. The PC map represents the first two principal components. The first PC incorporated about 85.93% of the total original variation, while the second accounted for 11.23%. The cluster pattern in the PC map, with high diversity in northern origin Chinese ethnic groups (except Tibetan), was in good agreement with the results of the NJ tree. The results are coincident with the geographic location and distribution of the language family. The first PC incorporated about 85.93% of the total original variation, while the second accounted for 11.23% DISCUSSION 1 Due to slipping of the DNA polymerase during replication (Malik et al., 2002a 2002a ; Lutz-Bonengel et al., 2004 2004 ) or nuclear factor dysregulation (Malik et al., 2002b 2002b ), the C-stretch exhibits significant variation among unrelated individuals. A recent study indicated that selective and neutral mechanisms might also be involved in the C-stretch length heteroplasmy in the mtDNA control region (Irwin et al., 2009 2009 ). In the current study, we determined the C-stretch haplotype profile of HVS-I in eight Chinese ethnic groups. The results indicate that the most common haplotype of HVS-I was AAAACCCCCTCCCC, and some novel haplotypes, population specific haplotypes, and novel mutations were also detected (Fig. A1 A1 ). The polymorphisms in HVS-I C-stretch resulted in relatively high values of gene diversity ( GD ), and low probability ( P ) values of two randomly selected individuals from a population having identical haplotype types in the eight Chinese ethnic groups. There was a significant correlation ( r= −0.961, P < 0.01) between the expansion of cytosine length and the reduction of upstream adenines in the C-stretch of HVS-I, supporting that sequence of cytosines vs adenosines was under some selective constraint (Howell and Smejkal, 2000 2000 ). These data provide further evidence that the C-stretch could be a useful genetic marker in forensic identification where genomic DNA is degraded or absent in a variety of forensic samples such as loose hairs, teeth or bone. However, in this study, mtDNA C-stretch length heteroplasmy was observed in the blood sample from selected Chinese populations, but its ratio was much lower than those in other populations (Chen et al., 2002 2002 ; Imaizumi et al., 2002 2002 ; Mabuchi et al., 2007 2007 ; Barbosa et al., 2008 2008 ). To ensure reproducible results, we used the average C-length of all clones as the representative of C-length for the samples with C-stretch length heteroplasmy. This method was certified to be useful in defining the actual C-length variant of C-stretch, as the average C-length gave us comprehensive information of all the different sequence-length variations from the same sample, rather than the limited information from the predominant sequence type (Table 2 2 ). The mtDNA nucleotide sequence evolves 6 to 17 times faster than nuclear DNA (Brown et al., 1982 1982 ), and the C-stretch of the HVS-I region evolves even faster than the other regions of mtDNA (Malik et al., 2002a 2002a ; Lutz-Bonengel et al., 2004 2004 ). We hypothesize that the mtDNA HVS-I C-stretch might therefore be useful as a marker in population genetics and molecular evolution studies of closely related populations, as its rapid rate of change can more accurately reveal more acute evolutionary events in recent human history. In this study, we chose eight closely related Chinese ethnic groups, including Northern Han, Southern Han, Tu, Tibetan, Salar, Yugur, Bonan, and Oroqen ethnic groups, to investigate the utility of the C-stretch genetic marker. These ethnic groups live very close to each other (Fig. 1 1 ), Northern Han (Xining and Xi’an), Tu, Tibetan, Salar, Yugur, Bonan ethnic groups live in Gansu and Qinghai Provinces, which are located in the northwest of China, and Oroqen and Han (Beijing) populations dwell in the northeast of China. All these ethnic groups belong to Sino-Tibetan or Altaic Language family (Table 1 1 ). The results from genetic matrix, phylogenetic tree, and PC map show highly coincident findings and indicate that the genetic relationships between the eight Chinese ethnic groups are very close, but relatively far from the Africans. A caveat here is that African populations are very diverse and the Africa sample data cited in this study were limited to certain regions of Africa (Kenya, Nigeria, and Niger). The Chinese ethnic minority groups, including Yugur, Bonan, Tu, and Sala, with the same language family, cluster together, while Tibetan ethnic group, which belongs to the Sino-Tibeta language family, was in another cluster. Ethnic groups within a similar geographic location (Yugur vs Bonan, Tu vs Salar) revealed a closer relationship. Therefore, the C-stretch variation is in agreement with the geographic location and distribution of the language family. These findings indicate that the C-stretch of mtDNA control region could be a reliable genetic marker when applied to population genetics and human evolution studies. A broad spectrum of degenerative diseases (Yen et al., 1989 1989 ; Cooper et al., 1992 1992 ; Bowling et al., 1993 1993 ; Brandon et al., 2005 2005 ) involving the central nervous system, heart, muscle, endocrine system, kidney and liver has been associated with mtDNA mutations, either base substitutions or insertion-deletions. The sequence-length variation of C-stretch may therefore affect mtDNA replication or transcription, and multiple diseases are associated with C-stretch variations (Tan et al., 2002 2002 ; Lee HC et al., 2004 2004 ; Meierhofer et al., 2004 2004 ; Montanini et al., 2005 2005 ; Sangkhathat et al., 2005 2005 ). Thereby, the mtDNA C-stretch might also be considered a useful biological marker for human disease. In this study, we identified three different novel mutations, which were T to A transversion at position 16 189, C to G transversion at position 16 184, and A to G transition at position 16 183. These novel mutations could be applied to a number of different research fields to further establish the relationships between mtDNA mutations and human disease. When using the direct sequencing approach, the ‘out-of-phase’ nucleotide pattern (Fig. 2 2 ), C-stretch could not easily be detected when compared to the other regions of mtDNA. Moreover, C-stretch length heteroplasmy further increases the difficulties of sequencing (Lee HY et al., 2004 2004 ; Lutz-Bonengel et al., 2004 2004 ; Bini and Pappalardo, 2005 2005 ). These obstacles might affect the application of mtDNA C-stretch in forensic identification, population genetics, and other studies. Several sensitive detection methods (Lee HY et al., 2004 2004 ; Lutz-Bonengel et al., 2004 2004 ), such as direct sequencing, fluorescently labeled restriction fragment analysis, cloning, and fragment analysis, have been reported. Of these methods, cloning is less likely to be affected by artifacts and shows the highest accuracy and sensitivity in defining the exact sequence variants. In this study, we directly sequenced all the samples at first. Unfortunately, some samples were no amenable to this procedure, in particularly, those with the influence of C-continuous tract or length heteroplasmy. 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Liver mitochondrial respiratory functions decline with age We thank Zhan-hai Wang of Forensic Medical Identification Center of Qinghai Public Security Bureau of China for his assistance of collecting samples. 10.1631/jzus.B0920140 Project supported by the Sciences and Technological Fundamental Resources Data of the Ministry of Education, China (No. 505015) and the Key Project for Science and Technology of Shaanxi Province, China (No. 2004K09-G12) 2009-05-11 2009-08-04 2009-07-08 2022-01-05 Copyright © 2009, Journal of Zhejiang University Science 2009 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 10 10