基于高通量测序技术研究成年大熊猫肠道菌群
|
摘要
选取6只成年健康大熊猫(Ailuropoda melanoleuca)、1只成年亚健康大熊猫,基于高通量测序技术,测定其肠道内细菌及真菌组成,研究性别和健康状况对成年大熊猫肠道微生物的影响,分析不同年龄段大熊猫肠道菌群的差异.结果表明,在门水平,成年大熊猫肠道内细菌主要为Proteobacteria和Firmicutes,真菌主要为Ascomycota和Basidiomycota.在属水平,成年大熊猫肠道细菌为Escherichia、Streptococcus、Clostridium及Leuconostoc,真菌为Humicola.雌性和雄性大熊猫肠道菌群没有显著性差异(P>0.05),健康大熊猫肠道细菌Esherichia、Clostridium(53.33%、1.10%)的含量比亚健康大熊猫(2.48%、0.00%)高,Streptococcus(22.88%)的含量比亚健康大熊猫(48.15%)低,健康大熊猫肠道真菌的多样性高于亚健康大熊猫.与文献比较发现,大熊猫肠道内菌群多样性为成年>老年>亚成体,随年龄增长,成年时Streptococcus(22.88%)、Leuconostoc(2.97%)和Clostridium(1.10%)的含量升高,Shigella(6.25%)和Citrobater(2.50%)的含量降低,老年时Weissella(16.67%)的含量升高,Leuconostoc(2.97%)和Clostridium(1.10%)的含量降低.本研究结果表明身体健康状况和年龄对大熊猫肠道微生物菌群结构组成有影响,成年大熊猫性别对肠道菌群没有影响.
To investigate the effects of sex, health condition, and age on the gut microbiomes of giant pandas, the structure and composition of bacteria and fungi of six adult giant pandas in health and one in sub-health were studied using high-throughput sequencing technology. Simultaneously, the gut microbiomes of giant pandas of various ages were compared based on previous studies. The gut microbiomes of female and male giant pandas were not significantly correlated(P > 0.05). The main bacterial phyla in healthy adult giant pandas consisted of Proteobacteria and Firmicutes, and the main fungal phyla consisted of Ascomycota and Basidiomycota. At the genus level, the bacteria mainly consisted of Escherichia, Streptococcus, Clostridium,and Leuconostoc, and the fungi mainly consisted of Humicola. However, the percentage of Esherichia and Clostridium(53.33% and 1.10%, respectively) in the intestinal flora and the diversity of fungi of giant pandas in health were both higher than those of giant pandas in sub-health, and the percentage of Streptococcus(22.88%) was lower. Comparing the gut microbiomes of subadult, adult, and geriatric giant pandas, the diversity in adult giant pandas was the highest, followed by that in geriatric and subadult pandas. The percentages of Streptococcus(22.88%), Clostridium(1.10%), and Leuconostoc(2.97%) in adult giant pandas were higher and those of Shigella(6.25%) and Citrobater(2.50%) were lower than those of sub-adult pandas. For geriatric giant pandas, the percentage of Weissella(16.67%) was higher and those of Leuconostoc(2.97%) and Clostridium(1.10%) were lower than those of adult giant pandas. Therefore, our study show that health condition and age probably have a significant influence on giant panda gut microbiomes while sex has not.
To investigate the effects of sex, health condition, and age on the gut microbiomes of giant pandas, the structure and composition of bacteria and fungi of six adult giant pandas in health and one in sub-health were studied using high-throughput sequencing technology. Simultaneously, the gut microbiomes of giant pandas of various ages were compared based on previous studies. The gut microbiomes of female and male giant pandas were not significantly correlated(P > 0.05). The main bacterial phyla in healthy adult giant pandas consisted of Proteobacteria and Firmicutes, and the main fungal phyla consisted of Ascomycota and Basidiomycota. At the genus level, the bacteria mainly consisted of Escherichia, Streptococcus, Clostridium,and Leuconostoc, and the fungi mainly consisted of Humicola. However, the percentage of Esherichia and Clostridium(53.33% and 1.10%, respectively) in the intestinal flora and the diversity of fungi of giant pandas in health were both higher than those of giant pandas in sub-health, and the percentage of Streptococcus(22.88%) was lower. Comparing the gut microbiomes of subadult, adult, and geriatric giant pandas, the diversity in adult giant pandas was the highest, followed by that in geriatric and subadult pandas. The percentages of Streptococcus(22.88%), Clostridium(1.10%), and Leuconostoc(2.97%) in adult giant pandas were higher and those of Shigella(6.25%) and Citrobater(2.50%) were lower than those of sub-adult pandas. For geriatric giant pandas, the percentage of Weissella(16.67%) was higher and those of Leuconostoc(2.97%) and Clostridium(1.10%) were lower than those of adult giant pandas. Therefore, our study show that health condition and age probably have a significant influence on giant panda gut microbiomes while sex has not.
引文
1 Meng QK,Miao F,Zhen J,Huang Y,Wang XY,Peng Y.Impact of earthquake-induced landslide on the habitat suitability of giant panda in Wolong,China[J].JMS,2016,13(10):1789-1805
2 鲍楠,刘成君,张和民,李德生.大熊猫肠道微生态的研究进展[J].畜牧与兽医,2005,37(4):57-59
3 鲍楠.野外放归大熊猫肠道菌群变化的研究[D].成都:四川大学,2006[Bao N.Studies on changes of the intestinal microf lora in giant panda returned to the wild[D].Chengdu:Sichuan University,2006]
4 Gevers D,Kugathasan S,Denson LA.The treatment-na?ve microbiome in new-onset Crohn’s disease[J].Cell Host Micr,2014,15(3):382-392
5 胡旭,王涛,梁姗,李薇,吴晓莉,金锋.肠道微生物与认知功能[J].中国微生态学杂志,2015,27(11):1359-1364[Hu X,Wang T,Liang S,Li W,Wu XL,Jin F.The role of gut microbiota in cognitive function[J].Chin J Microecol,2015,27(11):1359-1364]
6 刘广帅.雪豹肠道菌群多样性研究[D].山东:曲阜师范大学,2013[Liu GX.Studies of the intestinal microflora in snow leopard,2013]
7 杨伟平,王建刚,曹斌云.猪肠道微生物群落组成变化及其影响因素[J].中国畜牧杂志,2017,53(1):12-16[Yang WP,Wang JG,Cao BY.Composition and infuence factors of pigs’gut microbial community[J].Chin J Anim Sci,2017,53(1):12-16]
8 陈卫.肠道菌群:膳食与健康研究的新视角[J].食品科学技术学报,2015,33(6):1-6[Chen W.Gut microbiota—a new concern on health regulation[J].J Food Sci Technol,2015,33(6):1-6]
9 尹业师,王欣.影响实验小鼠肠道菌群的多因素比较研究[J].实验动物科学,2012,29(4):12-18[Yin YS,Wang X.Comparative study for factors that affect microbiota colonization in experimental mice[J].Lab Anim Sci,2012,29(4):12-18]
10 何廷美,崔婷婷,钟志军,王承东,何永果,王燚,张和民,李德生,李才武,张婷婷,罗永久,程安春,彭广能.成年大熊猫肠道菌群多样性的16S r DNA-RFLP分析[J].中国兽医科学,2012,42(11):1121-1127[He YM,Cui TT,Zhong ZJ,Wang CD,He YG,Wang Y,Zhang HM,Li DS,Li WC,Zhang TT,Luo YJ,Cheng AC,Peng GN.Research on diversity of intestinal flora in an adult giant panda base on the 16S r DNA-RFLP[J].Chin Vet Sci,2012,42(11):1121-1127]
11 李蓓,郭莉娟,龙梅,汤纯香,周晓平,罗燕,邹立扣.圈养大熊猫肠道微生物分离、鉴定及细菌耐药性研究[J].四川动物,2014,33(2):161-166[Li B,Guo LJ,Long M,Tang CX,Zhou XP,Luo Y,Zou LK.Isolation and antimicrobial resistance of bacteria from intestinal tract of giant panda[J].Sichuan J Zool,2014,33(2):161-166]
12 楼骏,柳勇,李延.高通量测序技术在土壤微生物多样性研究中的研究进展[J].中国农学通报,2014,30(15):256-260[Lou J,Liu Y,Li Y.Review of high-throughput sequencing techniques in studies of soil microbial diversity[J].Chin Agric Sci,2014,30(15):256-260]
13 曹荣,张井,孟辉辉,赵琳,刘淇.高通量测序与传统纯培养方法在牡蛎微生物群落分析中的应用对比[J].食品科学,2016,37(24):137-141[Cao R,Zhang J,Meng HH,Zhao L,Liu Q.Microbial Flora Analysis of Oyster:a comparison between traditional plate culture method and high throughput sequencing[J].Food Sci,2016,37(24):137-141]
14 陈蕾,周小理,周一鸣,李宗杰.高通量测序技术在肠道菌群研究中的应用[J].食品工业,2016,37(3):269-273[Chen L,Zhou XL,Zhou YM,Li ZJ.Application of high-throughput sequencing in intestinal microbiota[J].Food Ind,2016,37(3):269-273]
15 Mago?T,Salzberg SL.Flash:fast length ajustment of short reads to improve genome assemblies[J].Bioinformation,2011,27(21):2957-2963
16 Schnorr SL,Candela M,Rampelli S,Centan ni M,Consolandi C,Basaglia G,Turroni S,Biagi E,Peano C,Severgnini M,Fiori J,Gotti R,Bellis GD,Luiselli D,Brigidi P,Mabulla A,Marlowe F,Henry AG,Crittenden AN.Gut microbiome of the hadza hunter-gatherers[J].Nat Comm,2014,5(3654):3654
17 Caporaso JG,Kuczynski J,Stombaugh J,Bittinger K,Bittinger FD,Costello EK,Fierer N,Pe?a AG,Pe?a JK,Gordon JI,Huttley GA,Kelley ST,Knights D,Koenig JE,Ley RE,Lozupone GA,Mcdonald D,Muegge BD,Pir r ung M,Reeder J,Sevinsky JR,Tur nbaugh PJ,Walters WA,Widmann J,Yatsunenko T,Yatsunenko J,Knight R.Qiime allows analysis of high-throughput community sequencing data[J].Nat Methods,2010,7(5):335-336
18 Edgar RC,Haas BJ,Clemente JC,Quince C,Knight R.Uchime improves sensitivity and speed of chimera detection[J].Bioinformation,2011,27(16):2194-2200
19 Pitta DW,Parmar N,Patel AK,Indugu N,Prajapathi KB,Patel AB,Reddy B,Joshi C.Bacterial diversity dynamics associated with different diets and different primer pairs in the rumen of kankrej cattle[J].PLo S ONE,2014,9(11):e111710
20 张志和,张安居.大熊猫肠道正常菌群的研究[J].兽类学报,1995,15(3):170-175
21 王晓艳.成年与老年大熊猫肠道菌群16Sr DNA-RFLP技术分析[D].雅安:四川农业大学,2013[Wang XY.Analysis the intestinal flora of adult and elderly panda by 16S r DNA-RFLP technology[D].Ya’an:Sichuan Agric University,2013]
22 王立志,徐谊英.圈养大熊猫粪便中微生物多样性的研究[J].四川动物,2016,35(1):17-23[Wang LZ,Xu YY.Diversity of microorganism in the feces of captive giant pandas[J].Sichuan J Zool,2016,35(1):17-23].
23 周紫峣,钟志军,周潇潇,彭广能,李奇,陶志永,杨平,杨俊,陈英,王亚萍,谭和林.大熊猫肠道菌群的研究进展[J].微生物学通报,2016,43(6):1366-71[Zhou ZX,Zhong ZJ,Zhou XX,Peng GN,Li Q,Tao ZY,Yang P,Yang J,Chen Y,Wang YP,Tan HL.Giant panda intestinal bacteria-a review[J].Microbiol China,2016,43(6):1366-1371]
24 王勇,邹秉杰,刘云龙,李思勤,周国华.不同粪便DNA提取试剂盒及样本储存时间对粪便人基因组DNA提取率的影响[J].临床误诊误治,2014,27(4):77-80[Wang Y,Zou BJ,Liu YL,Li SQ,Zhou GH.Inf luence of different storage times and DNAzol reagent kits on the efficiency of human genomic DNA extraction from stools[J].Clin Misd Misth,2014,27(4):77-80]
25 艾生权.亚成体大熊猫肠道真菌多样性的初步研究[D].雅安:四川农业大学,2014[Ai SQ.Preliminary study of fungi diversity in intestines of subadult giant panda[D].Ya’an:Sichuan Agric University,2014]
26 艾生权,钟志军,彭广能,王承东,罗永久,何延美,谷武阳,李才武,李刚诗,吴虹林,刘学涵,夏玉,刘艳红,周潇潇.亚成体大熊猫肠道真菌多样性[J].微生物学报,2014,54(11):1344-1352[Ai SQ,Zhong ZJ,Peng GN,Wang CD,Lou YJ,He YM,Gu WY,Li CW,Li GS,Wu HL,Liu XH,Xia Y,Liu YH,Zhon XX.Intestinal fungal diversity of sub-adult giant panda.Acta Microbiol Sin,2014,54(1):1344-1352]
27 胡锦矗.从野外大熊猫的粪便估计年龄及其种群年龄结构的研究[J].兽类学报,1987,7(2):81-84
28 Xue ZS,Zhang WP,Wang LH,Hou R,Zhang MH,Fei LS,Zhang XJ,Huang H,Bridgewater LC,Jiang Y,Jiang CL,Zhao LP,Pang XY,Zhang ZH.The bamboo-eating giant panda harbors a carnivore-like gut microbiota,with excessive seasonal variations[J].MBIO,2015,6(3):22-15
29 袁施彬,屈元元,张泽钧,赵金刚,侯蓉,王海瑞.圈养大熊猫食谱组成与营养成分分析[J].兽类学报,2015,35(1):65-73[Yuan SB,Qu YY,Zhang ZJ,Zhao JG,Hou R,Wang HR.Composition and nutrient analysis of captive giant panda diet[J].Acta Theriol Sin,2015,35(1):65-73]
30 王海娟,潘渠.大熊猫肠道正常菌群降解纤维素的机制[J].中国微生态学杂志,2014,26(2):225-228[Wang HJ,Pan Q.The mechanism of cellulose metabolism by the giant panda gut microbiome[J].Chin J Microecol,2014,26(2):225-228]
30 刘选珍,李明喜,胡建秋,黄大明,杨智,黄详明.圈养亚成体大熊猫日粮粗蛋白表观消化率的初步研究[J].应用与环境生物学报,2002,8(2):174-178[Liu XZ,Li MX,Hu JQ,Huang DM,Yang Z,Huang XM.Study on apparent digestibility of crude protein by captive sub-adult giant panda[J].Chin J Appl Environ Biol,2002,8(2):174-178
32 王燚.基于ERIC-PCR和16S r DNA-RFLP技术对亚成体大熊猫肠道菌群结构的研究[D].雅安:四川农业大学,2011[Wang Y.Estimation of giant panda’s intestinal f lora structure in intestine based on 16S r DNA-RFLP technologies[D].Ya’an:Sichuan Agriculture University,2011]
33 王晓艳,袁听,廖虹,罗永久,何延美,王承东,刘小敏,吴虹林,彭广能.圈养老年大熊猫肠道菌群16S r DNA克隆文库的建立[J].中国畜牧兽医,2015,42(6):1402-1408[Wang XY,Yuan T,Liao H,Luo YJ,He YM,Wang CD,Liu XM,Wu HL,Peng GN.Construction of the 16S r DNA clone library of intestinal flora of captive elderly giant panda[J].Anim Husb Vet Med,2015,42(6):1402-1408]
2 鲍楠,刘成君,张和民,李德生.大熊猫肠道微生态的研究进展[J].畜牧与兽医,2005,37(4):57-59
3 鲍楠.野外放归大熊猫肠道菌群变化的研究[D].成都:四川大学,2006[Bao N.Studies on changes of the intestinal microf lora in giant panda returned to the wild[D].Chengdu:Sichuan University,2006]
4 Gevers D,Kugathasan S,Denson LA.The treatment-na?ve microbiome in new-onset Crohn’s disease[J].Cell Host Micr,2014,15(3):382-392
5 胡旭,王涛,梁姗,李薇,吴晓莉,金锋.肠道微生物与认知功能[J].中国微生态学杂志,2015,27(11):1359-1364[Hu X,Wang T,Liang S,Li W,Wu XL,Jin F.The role of gut microbiota in cognitive function[J].Chin J Microecol,2015,27(11):1359-1364]
6 刘广帅.雪豹肠道菌群多样性研究[D].山东:曲阜师范大学,2013[Liu GX.Studies of the intestinal microflora in snow leopard,2013]
7 杨伟平,王建刚,曹斌云.猪肠道微生物群落组成变化及其影响因素[J].中国畜牧杂志,2017,53(1):12-16[Yang WP,Wang JG,Cao BY.Composition and infuence factors of pigs’gut microbial community[J].Chin J Anim Sci,2017,53(1):12-16]
8 陈卫.肠道菌群:膳食与健康研究的新视角[J].食品科学技术学报,2015,33(6):1-6[Chen W.Gut microbiota—a new concern on health regulation[J].J Food Sci Technol,2015,33(6):1-6]
9 尹业师,王欣.影响实验小鼠肠道菌群的多因素比较研究[J].实验动物科学,2012,29(4):12-18[Yin YS,Wang X.Comparative study for factors that affect microbiota colonization in experimental mice[J].Lab Anim Sci,2012,29(4):12-18]
10 何廷美,崔婷婷,钟志军,王承东,何永果,王燚,张和民,李德生,李才武,张婷婷,罗永久,程安春,彭广能.成年大熊猫肠道菌群多样性的16S r DNA-RFLP分析[J].中国兽医科学,2012,42(11):1121-1127[He YM,Cui TT,Zhong ZJ,Wang CD,He YG,Wang Y,Zhang HM,Li DS,Li WC,Zhang TT,Luo YJ,Cheng AC,Peng GN.Research on diversity of intestinal flora in an adult giant panda base on the 16S r DNA-RFLP[J].Chin Vet Sci,2012,42(11):1121-1127]
11 李蓓,郭莉娟,龙梅,汤纯香,周晓平,罗燕,邹立扣.圈养大熊猫肠道微生物分离、鉴定及细菌耐药性研究[J].四川动物,2014,33(2):161-166[Li B,Guo LJ,Long M,Tang CX,Zhou XP,Luo Y,Zou LK.Isolation and antimicrobial resistance of bacteria from intestinal tract of giant panda[J].Sichuan J Zool,2014,33(2):161-166]
12 楼骏,柳勇,李延.高通量测序技术在土壤微生物多样性研究中的研究进展[J].中国农学通报,2014,30(15):256-260[Lou J,Liu Y,Li Y.Review of high-throughput sequencing techniques in studies of soil microbial diversity[J].Chin Agric Sci,2014,30(15):256-260]
13 曹荣,张井,孟辉辉,赵琳,刘淇.高通量测序与传统纯培养方法在牡蛎微生物群落分析中的应用对比[J].食品科学,2016,37(24):137-141[Cao R,Zhang J,Meng HH,Zhao L,Liu Q.Microbial Flora Analysis of Oyster:a comparison between traditional plate culture method and high throughput sequencing[J].Food Sci,2016,37(24):137-141]
14 陈蕾,周小理,周一鸣,李宗杰.高通量测序技术在肠道菌群研究中的应用[J].食品工业,2016,37(3):269-273[Chen L,Zhou XL,Zhou YM,Li ZJ.Application of high-throughput sequencing in intestinal microbiota[J].Food Ind,2016,37(3):269-273]
15 Mago?T,Salzberg SL.Flash:fast length ajustment of short reads to improve genome assemblies[J].Bioinformation,2011,27(21):2957-2963
16 Schnorr SL,Candela M,Rampelli S,Centan ni M,Consolandi C,Basaglia G,Turroni S,Biagi E,Peano C,Severgnini M,Fiori J,Gotti R,Bellis GD,Luiselli D,Brigidi P,Mabulla A,Marlowe F,Henry AG,Crittenden AN.Gut microbiome of the hadza hunter-gatherers[J].Nat Comm,2014,5(3654):3654
17 Caporaso JG,Kuczynski J,Stombaugh J,Bittinger K,Bittinger FD,Costello EK,Fierer N,Pe?a AG,Pe?a JK,Gordon JI,Huttley GA,Kelley ST,Knights D,Koenig JE,Ley RE,Lozupone GA,Mcdonald D,Muegge BD,Pir r ung M,Reeder J,Sevinsky JR,Tur nbaugh PJ,Walters WA,Widmann J,Yatsunenko T,Yatsunenko J,Knight R.Qiime allows analysis of high-throughput community sequencing data[J].Nat Methods,2010,7(5):335-336
18 Edgar RC,Haas BJ,Clemente JC,Quince C,Knight R.Uchime improves sensitivity and speed of chimera detection[J].Bioinformation,2011,27(16):2194-2200
19 Pitta DW,Parmar N,Patel AK,Indugu N,Prajapathi KB,Patel AB,Reddy B,Joshi C.Bacterial diversity dynamics associated with different diets and different primer pairs in the rumen of kankrej cattle[J].PLo S ONE,2014,9(11):e111710
20 张志和,张安居.大熊猫肠道正常菌群的研究[J].兽类学报,1995,15(3):170-175
21 王晓艳.成年与老年大熊猫肠道菌群16Sr DNA-RFLP技术分析[D].雅安:四川农业大学,2013[Wang XY.Analysis the intestinal flora of adult and elderly panda by 16S r DNA-RFLP technology[D].Ya’an:Sichuan Agric University,2013]
22 王立志,徐谊英.圈养大熊猫粪便中微生物多样性的研究[J].四川动物,2016,35(1):17-23[Wang LZ,Xu YY.Diversity of microorganism in the feces of captive giant pandas[J].Sichuan J Zool,2016,35(1):17-23].
23 周紫峣,钟志军,周潇潇,彭广能,李奇,陶志永,杨平,杨俊,陈英,王亚萍,谭和林.大熊猫肠道菌群的研究进展[J].微生物学通报,2016,43(6):1366-71[Zhou ZX,Zhong ZJ,Zhou XX,Peng GN,Li Q,Tao ZY,Yang P,Yang J,Chen Y,Wang YP,Tan HL.Giant panda intestinal bacteria-a review[J].Microbiol China,2016,43(6):1366-1371]
24 王勇,邹秉杰,刘云龙,李思勤,周国华.不同粪便DNA提取试剂盒及样本储存时间对粪便人基因组DNA提取率的影响[J].临床误诊误治,2014,27(4):77-80[Wang Y,Zou BJ,Liu YL,Li SQ,Zhou GH.Inf luence of different storage times and DNAzol reagent kits on the efficiency of human genomic DNA extraction from stools[J].Clin Misd Misth,2014,27(4):77-80]
25 艾生权.亚成体大熊猫肠道真菌多样性的初步研究[D].雅安:四川农业大学,2014[Ai SQ.Preliminary study of fungi diversity in intestines of subadult giant panda[D].Ya’an:Sichuan Agric University,2014]
26 艾生权,钟志军,彭广能,王承东,罗永久,何延美,谷武阳,李才武,李刚诗,吴虹林,刘学涵,夏玉,刘艳红,周潇潇.亚成体大熊猫肠道真菌多样性[J].微生物学报,2014,54(11):1344-1352[Ai SQ,Zhong ZJ,Peng GN,Wang CD,Lou YJ,He YM,Gu WY,Li CW,Li GS,Wu HL,Liu XH,Xia Y,Liu YH,Zhon XX.Intestinal fungal diversity of sub-adult giant panda.Acta Microbiol Sin,2014,54(1):1344-1352]
27 胡锦矗.从野外大熊猫的粪便估计年龄及其种群年龄结构的研究[J].兽类学报,1987,7(2):81-84
28 Xue ZS,Zhang WP,Wang LH,Hou R,Zhang MH,Fei LS,Zhang XJ,Huang H,Bridgewater LC,Jiang Y,Jiang CL,Zhao LP,Pang XY,Zhang ZH.The bamboo-eating giant panda harbors a carnivore-like gut microbiota,with excessive seasonal variations[J].MBIO,2015,6(3):22-15
29 袁施彬,屈元元,张泽钧,赵金刚,侯蓉,王海瑞.圈养大熊猫食谱组成与营养成分分析[J].兽类学报,2015,35(1):65-73[Yuan SB,Qu YY,Zhang ZJ,Zhao JG,Hou R,Wang HR.Composition and nutrient analysis of captive giant panda diet[J].Acta Theriol Sin,2015,35(1):65-73]
30 王海娟,潘渠.大熊猫肠道正常菌群降解纤维素的机制[J].中国微生态学杂志,2014,26(2):225-228[Wang HJ,Pan Q.The mechanism of cellulose metabolism by the giant panda gut microbiome[J].Chin J Microecol,2014,26(2):225-228]
30 刘选珍,李明喜,胡建秋,黄大明,杨智,黄详明.圈养亚成体大熊猫日粮粗蛋白表观消化率的初步研究[J].应用与环境生物学报,2002,8(2):174-178[Liu XZ,Li MX,Hu JQ,Huang DM,Yang Z,Huang XM.Study on apparent digestibility of crude protein by captive sub-adult giant panda[J].Chin J Appl Environ Biol,2002,8(2):174-178
32 王燚.基于ERIC-PCR和16S r DNA-RFLP技术对亚成体大熊猫肠道菌群结构的研究[D].雅安:四川农业大学,2011[Wang Y.Estimation of giant panda’s intestinal f lora structure in intestine based on 16S r DNA-RFLP technologies[D].Ya’an:Sichuan Agriculture University,2011]
33 王晓艳,袁听,廖虹,罗永久,何延美,王承东,刘小敏,吴虹林,彭广能.圈养老年大熊猫肠道菌群16S r DNA克隆文库的建立[J].中国畜牧兽医,2015,42(6):1402-1408[Wang XY,Yuan T,Liao H,Luo YJ,He YM,Wang CD,Liu XM,Wu HL,Peng GN.Construction of the 16S r DNA clone library of intestinal flora of captive elderly giant panda[J].Anim Husb Vet Med,2015,42(6):1402-1408]