Breeding and Propagation of FAT1 Transgenic Ancestral Bull

doi:10.13304/j.nykjdb.2021.0498

Abstract

Abstract:

n-3 polyunsaturated fatty acids （n-3 PUFAs） are essential fatty acids for human health and can inhibit many diseases. Using transgenic technology to produce FAT1 transgenic cattle can improve the content of n-3 PUFAs in beef and milk， and provide healthy food for human. In this study， after propagation of FAT1 transgenic cattle through superovulation and embryo transfer （MOET） technique， the growth index and genetic stability of F₁ generation cattle were tested. After that， ZK005 bull was determined as ancestry based on the analysis. The F₂ generation was produced by artificial insemination with frozen semen of ZK005， and its growth indexes， genetic stability and blood biochemical indexes were measured. The results showed that 8 FAT1 gene positive F₁ cattle were obtained. There was no significant difference in growth index between F₁ and wild-type cattle. The content of n-3 PUFAs increased significantly and the ratio of n-6/n-3 PUFAs decreased significantly. The content of n-3 PUFAs in various tissues significantly increased， especially in muscles. Total of 12 F₂ generation cattle， were obtained by artificial insemination. There was no significant difference in growth and development and blood biochemical indexes between F₂ generation cattle and wild-type cattle as a whole， while the above research showed that breeding FAT1 transgenic cattle with transgenic technology could cultivate new transgenic cattle strains （species） rich in n-3 PUFAs， improved the content of n-3 PUFAs in beef and milk， and FAT1 gene could be stably and effectively expressed in F₁ and F₂ generation cattle， which had no adverse impact on the health of transgenic cattle and could provide more nutritious food for human beings.

Key words: n-3 polyunsaturated fatty acids, ancestral bull, genetic stability, blood biochemical indexes

摘要：

n-3多不饱和脂肪酸（n-3 polyunsaturated fatty acids，n-3 PUFAs）是人体必需脂肪酸，对多种疾病有抑制作用。利用超数排卵与胚胎移植技术（superovulation and embryo transfer，MOET）对FAT1转基因牛进行了扩繁，并对F₁代牛进行了生长指标测定与遗传稳定性分析，在此基础上确定了系祖公牛ZK005；用ZK005的冻精经人工授精生产F₂代，并对其进行生长指标测定、遗传稳定性分析和血液生化指标检测。结果显示，原代牛的MOET扩繁效果良好，获得了8头FAT1基因阳性F₁代牛；F₁代牛生长指标与野生型牛无显著差异；外源基因稳定整合，n-3 PUFAs含量显著提高，n-6多不饱和脂肪酸/n-3多不饱和脂肪酸（n-6 polyunsaturated fatty acids/n-3 polyunsaturated fatty acids，n-6/n-3）比值显著降低；F₁代牛各组织中n-3 PUFAs含量显著提高，其中肌肉中提高最明显。从F₁代牛中选择备用系祖公牛ZK005，其精液冷冻后活力达40%，经人工输精获得F₂代牛12头，F₂代牛生长发育与血液生化指标整体上与野生型牛无显著差异。以上研究表明，利用转基因技术选育FAT1转基因牛，可培育富含n-3 PUFAs的转基因牛新品系（种），提高牛肉、奶中n-3 PUFAs含量，且FAT1基因在F₁和F₂代牛中均能稳定有效表达，对转基因牛的健康无不利影响，可为人类提供更营养的食品。

关键词: n-3多不饱和脂肪酸, 系祖, 遗传稳定性, 血液生化指标

CLC Number:

S823

Chunling BAI, Lei YANG, Guanghua SU, Zhuying WEI, Xueqiao WANG, Lin ZHU, Xinyu ZHOU, Chao HAI, Di WU, Xuefei LIU, Yunxi WU, Li ZHANG, Guangpeng LI. Breeding and Propagation of FAT1 Transgenic Ancestral Bull[J]. Journal of Agricultural Science and Technology, 2022, 24(9): 66-78.

白春玲, 杨磊, 苏广华, 魏著英, 王学侨, 朱琳, 周新宇, 海超, 吴迪, 刘雪霏, 武云喜, 张立, 李光鹏. FAT1转基因系祖公牛选育及其扩繁研究[J]. 中国农业科技导报, 2022, 24(9): 66-78.

Figures/Tables 10

References 43

1	RUSSO G L. Dietary n-6 and n-3 polyunsaturated fatty acids: from biochemistry to clinical implications in cardiovascular prevention [J]. Biochem. Pharmacol., 2009, 77(6): 937-946.
2	GRIFFIN B A. How relevant is the ratio of dietary n-6 to n-3 polyunsaturated fatty acids to cardiovascular disease risk? evidence from the OPTILIP study [J]. Curr. Opin. Lipidol., 2008, 19(1): 57-62.
3	SIMOPOULOS A P. An increase in the omega-6/omega-3 fatty acid ratio increases the risk for obesity [J/OL]. Nutrients, 2016, 8(3):128 [2022-04-24]. .
4	GOODSTINE S L, ZHENG T, HOLFORD T R, et al.. Dietary (n-3)/(n-6) fatty acid ratio: possible relationship to premenopausal but not postmenopausal breast cancer risk in US women [J]. J. Nutr., 2003, 133(5): 1409-1414.
5	WILLIAMS C D, WHITLEY B M, HOYO C, et al.. A high ratio of dietary n-6/n-3 polyunsaturated fatty acids is associated with increased risk of prostate cancer [J]. Nutr. Res., 2011, 31(1): 1-8.
6	ZHU Z J, JIANG W Q, MCGINLEY J N, et al.. Mammary gland density predicts the cancer inhibitory activity of the n-3 to n-6 ratio of dietary FAT [J]. Cancer Prev. Res., 2011, 4(10): 1675-1685.
7	KALOGEROPOULOS N, PANAGIOTAKOS D B, PITSAVOS C, et al.. Unsaturated fatty acids are inversely associated and n-6/n-3 ratios are positively related to inflammation and coagulation markers in plasma of apparently healthy adults [J]. Clin. Chim. Acta., 2010, 411(7-8): 584-591.
8	JUMPSEN J, LIEN E L, GOH Y K, et al.. Small changes of dietary (n-6) and (n-3)/fatty acid content ration alter phosphatidylethanolamine and phosphatidylcholine fatty acid composition during development of neuronal and glial cells in rats [J]. J. Nutr., 1997, 127(5): 724-731.
9	SANTILLÁN M E, VINCENTI L M, MARTINI A C, et al.. Developmental and neurobehavioral effects of perinatal exposure to diets with different omega-6:omega-3 ratios in mice [J]. Nutrition, 2010, 26(4): 423-431.
10	SIMOPOULOS A P. Importance of the ratio of omega-6/omega-3 essential fatty acids: evolutionary aspects [J]. World Rev. Nutr. Diet., 2003, 92: 1-22.
11	HUANG Q, WEN J, CHEN G, et al.. Omega-3 polyunsaturated fatty acids inhibited tumor growth via preventing the decrease of genomic DNA methylation in colorectal cancer rats [J]. Nutr. Cancer, 2016, 68(1) :113-119.
12	KANG J X. From fat to fat-1: a tale of omega-3 fatty acids [J]. J. Membr. Biol., 2005, 206(2): 165-172.
13	SIMOPOULOS A P. The importance of the omega-6/omega-3 fatty acid ratio in cardiovascular disease and other chronic diseases [J]. Exp. Biol. Med. (Maywood), 2008, 233(6): 674-688.
14	LESA G M, PALFREYMAN M, HALL D H, et al.. Long chain polyunsaturated fatty acids are required for efficient neurotransmission in C. elegans [J]. J. Cell Sci., 2003, 116(24): 4965-4975.
15	YOSHIDA K, HASHIMOTO M, HORI R, et al.. Bacterial long-chain polyunsaturated fatty acids: their biosynthetic genes, functions, and practical use [J/OL]. Mar. Drugs, 2016, 14(5): 94[2022-05-10]. .
16	KHOZIN-GOLDBERG I, ISKANDAROV U, COHEN Z. LC-PUFA from photosynthetic microalgae: occurrence, biosynthesis, and prospects in biotechnology [J]. Appl. Microbiol. Biotechnol., 2011, 91(4): 905-915.
17	LAI L X, KANG J X, LI R F, et al.. Generation of cloned transgenic pigs rich in omega-3 fatty acids [J]. Na Biotechnol., 2006, 24(4): 435-436.
18	PAN D, ZHANG L, ZHOU Y, et al.. Efficient production of omega-3 fatty acid desaturase (sFAT1)-transgenic pigs by somatic cell nuclear transfer [J]. Sci. China Life Sci., 2010, 53(4): 517-523.
19	TANG M, ZHENG X, CHENG W, et al.. Safety assessment of sFAT1 transgenic pigs by detecting their co-habitant microbe in intestinal tract [J]. Transgenic Res., 2011, 20(4): 749-758.
20	ZHANG P, ZHANG Y, DOU H, et al.. Handmade cloned transgenic piglets expressing the nematode FAT1 gene [J]. Cell Reprogram., 2012, 14(3): 258-266.
21	ZHOU Y, LIN Y, WU X, et al.. The high-level accumulation of n-3 polyunsaturated fatty acids in transgenic pigs harboring the n-3 fatty acid desaturase gene from Caenorhabditis briggsae [J]. Transgenic Res., 2014, 23(1): 89-97.
22	LI M, OUYANG H, YUAN H, et al.. Site-specific FAT1 knock-in enables significant decrease of n-6 PUFAs/n-3 PUFAs ratio in pigs [J]. G3 (Bethesda), 2018, 8(5): 1747-1754.
23	TANG F, YANG X, LIU D, et al.. Co-expression of FAT1 and FAT2 in transgenic pigs promotes synthesis of polyunsaturated fatty acids [J]. Transgenic Res., 2019, 28(3-4): 369-379.
24	WU X, OUYANG H, DUAN B, et al.. Production of cloned transgenic cow expressing omega-3 fatty acids [J]. Transgenic Res., 2012, 21(3): 537-543.
25	王丙萍,刘羿羿,王峰,等.体细胞核移植生产转FAT1基因克隆Holstein牛[J].中国兽医学报,2015,35(7):1199-1204.
	WANG B P, LIU Y Y, WANG F, et al.. The cloned holstein cows derived from FAT1 gene transferred somatic cell [J]. Chin. J. Vet. Sci., 2015, 35(7): 1199-1204.
26	CHENG G, FU C, WANG H, et al.. Production of transgenic beef cattle rich in n-3 PUFAs by somatic cell nuclear transfer [J]. Biotechnol. Lett., 2015, 37(8): 1565-1571.
27	段彪,程磊,苏广华,等.ω-3转基因克隆绵羊诞生[J]. 内蒙古大学学报, 2011, 42(1):121-122.
	DUAN B, CHENG L, SU G H, et al.. ω-transgenic cloned sheep production[J]. J. Inner Mongolia Univ., 2011, 42(1): 121-122.
28	ZHANG P, LIU P, DOU H, et al.. Handmade cloned transgenic sheep rich in omega-3 fatty acids [J/OL]. PLoS One, 2013, 8(2): e55941 [2022-04-24]. .
29	宋丽爽,高广琦,魏著英,等. FAT1转基因牛奶对小鼠健康和生殖能力的影响[J].实验动物科学,2017,34(3): 28-33, 37.
	SONG L S, GAO G Q, WEI Z Y, et al.. The health and reproduction of the mice fed by diets supplemented with milk from FAT1 transgenic cow [J]. Lab. Anim. Sci., 2017, 34(3) :28-33, 37.
30	许智宏,李晨.我国转基因生物产业化亟待突破[N].中国科学报,2021-03-18(001).
31	YAN C, DING X, DASGUPTA N, et al.. Gene profile of myeloid-derived suppressive cells from the bone marrow of lysosomal acid lipase knock-out mice [OL]. PLoS One, 2012, 7(2): e30701 [2022-04-24]. .
32	于超然. 转FAT1基因牛外源基因整合位点鉴定[D].呼和浩特:内蒙古大学,2014.
	YU C R. Identification of the transgenic integration site in FAT1 transgenic cattle [D]. Hohhot: Inner Mougolia University, 2014.
33	华文君,刘西梅,程妮,等.转sFat1-基因猪生理生化指标与繁殖性能测定[J].安徽农业科学,2010,38(32):18235-18236.
	HUA W J, LIU X M, CHENG N, et al.. Determination of physiological biochemical parameters and reproductive performance in transgenic FAT1 gene pigs [J]. J. Anhui Agric. Sci., 2010, 38(32): 18235-18236.
34	RAHEJA B S, SADIKOT S M, PHATAK R B, et al.. Significance of the N-6/N-3 ratio for insulin action in diabetes [J]. Ann. NY Acad. Sci., 1993, 683: 258-271
35	KUDA O, JELENIK T, JILKOVA Z, et al.. N-3 fatty acids and rosiglitazone improve insulin sensitivity through additive stimulatory effects on muscle glycogen synthesis in mice fed a high-FAT diet [J]. Diabetologia, 2009, 52(5): 941-951.
36	LI N, YUE H, JIA M, et al.. Effect of low-ratio n-6/n-3 PUFA on blood glucose: a meta-analysis [J]. Food Funct., 2019, 10(8): 4557-4565.
37	SMITH B K, HOLLOWAY G P, REZA-LOPEZ S, et al.. A decreased n-6/n-3 ratio in the FAT1 mouse is associated with improved glucose tolerance [J]. Appl. Physiol. Nutr. Metab., 2010, 35(5): 699-706.
38	KIM E H, BAE J S, HAHM K B, et al.. Endogenously synthesized n-3 polyunsaturated fatty acids in FAT1 mice ameliorate high-FAT diet-induced non-alcoholic fatty liver disease [J]. Biochem. Pharmacol., 2012, 84(10): 1359-1365.
39	IKEDA I, WAKAMATSU K, INAYOSHI A, et al.. alpha-Linolenic, eicosapentaenoic and docosahexaenoic acids affect lipid metabolism differently in rats [J]. J. Nutr., 1994, 124(10): 1898-1906.
40	CONQUER J A, HOLUB B J. Effect of supplementation with different doses of DHA on the levels of circulating DHA as non-esterified fatty acid in subjects of Asian Indian background [J]. J. Lipid Res., 1998, 39(2): 286-292.
41	SKULAS-RAY A C, WILSON P W F, HARRIS W S, et al.. Omega-3 fatty acids for the management of hypertriglyceridemia: a science advisory from the American Heart Association [J/OL]. Circulation, 2019, 140(12): e673-e691 [2022-04-24]. .
42	罗娜,孙志宏. Omega-3多不饱和脂肪酸在高脂血症中的作用机制研究进展[J].中国油脂,2020,45(7):97-101.
	LUO N, SUN Z H. Progress in action mechanism of omega-3 polyunsaturated fatty acids in hyperlipidemia [J]. China Oils Fats, 2020, 45(7): 97-101.
43	李新萍,陶岳,张孝恩,等.新疆石河子地区奶牛血液生化指标正常参考值范围的建立[J].中国奶牛,2011(18):47-50.
	LI X P, TAO Y, ZHANG X E, et al.. Establishment of the normal reference range of blood biochemical parameters for dairy cow in Shihezi area of Xinjiang [J]. China Dairy Cattle, 2011(18): 47-50.

类型	编号 Number	目的基因 Target gene	n-6 PUFAs/%	n-3 PUFAs/%	n-6/n-3 PUFAs	n-6/n-3 PUFAs平均值 n-6/n-3 PUFAs average value
FAT1基因阳性牛 FAT1 gene positive cattle	ZK004	+	2.08	1.86	1.12	3.93 b
	ZK005	+	7.69	3.35	2.29
	FD001	+	18.44	8.50	2.17
	FD002	+	37.15	4.65	7.98
	FD003	+	19.77	2.36	8.38
	FD004	+	14.08	5.31	2.65
	FD005	+	12.11	2.93	4.13
	FD006	+	9.86	3.62	2.72
野生型牛 Wild type cattle	WT-1	NA	1.89	0.12	15.75	16.10 a
	WT-2	NA	4.97	0.47	10.57
	WT-3	NA	11.21	0.51	21.98
FAT1基因阴性牛 FAT1 gene negative cattle	Con 1	-	18.11	1.71	10.59	13.14 a
	Con 2	-	4.90	0.38	12.90
	Con 3	-	21.99	1.38	15.93

类型	编号 Number	目的基因 Target gene	n-6 PUFAs/%	n-3 PUFAs/%	n-6/n-3 PUFAs	n-6/n-3 PUFAs平均值 n-6/n-3 PUFAs average value
FAT1基因阳性牛 FAT1 gene positive cattle	ZK004	+	2.08	1.86	1.12	3.93 b
	ZK005	+	7.69	3.35	2.29
	FD001	+	18.44	8.50	2.17
	FD002	+	37.15	4.65	7.98
	FD003	+	19.77	2.36	8.38
	FD004	+	14.08	5.31	2.65
	FD005	+	12.11	2.93	4.13
	FD006	+	9.86	3.62	2.72
野生型牛 Wild type cattle	WT-1	NA	1.89	0.12	15.75	16.10 a
	WT-2	NA	4.97	0.47	10.57
	WT-3	NA	11.21	0.51	21.98
FAT1基因阴性牛 FAT1 gene negative cattle	Con 1	-	18.11	1.71	10.59	13.14 a
	Con 2	-	4.90	0.38	12.90
	Con 3	-	21.99	1.38	15.93

性别 Sex	月龄/月 Month age/mouth	体质量 Body mass/kg		体高 Body height/cm		十字部宽 Height at hip cross/cm		胸围 Bust/cm
性别 Sex	月龄/月 Month age/mouth	转基因组 Transgenic	对照组 Control	转基因组 Transgenic	对照组 Control	转基因组 Transgenic	对照组 Control	转基因组 Transgenic	对照组 Control
母牛Cow	0	28.80	27.48	79.22	79.00	17.18	17.20	72.78	73.04
	3	125.80	115.40	92.22	91.11	24.82	22.88	110.95	110.44
	6	174.45	179.60	99.82	99.74	28.14	27.56	124.90	123.13
	9	218.60	225.15	106.23	106.56	30.45	31.06	136.50	134.70
	12	256.70	248.70	117.06	117.86	35.52	35.45	151.62	153.02
	15	271.20	275.60	122.52	121.79	36.04	36.22	157.53	155.44
	18	373.90	386.25	124.98	125.74	37.84	37.80	167.14	165.13
公牛Bull	0	30.60	28.70	80.00	79.70	17.50	17.40	73.40	73.20
	3	131.40	120.40	92.60	93.05	24.50	25.20	113.25	113.10
	6	201.35	192.20	102.40	102.00	29.20	28.10	126.30	125.45
	9	249.60	248.05	108.15	107.30	32.05	32.20	138.10	137.50
	12	297.10	292.50	118.20	117.80	36.10	35.85	154.00	154.20
	15	332.00	335.40	124.30	123.95	37.50	36.90	159.85	158.10
	18	443.70	456.35	127.20	126.20	39.60	39.50	168.60	168.25

性别 Sex	月龄/月 Month age/mouth	体质量 Body mass/kg		体高 Body height/cm		十字部宽 Height at hip cross/cm		胸围 Bust/cm
性别 Sex	月龄/月 Month age/mouth	转基因组 Transgenic	对照组 Control	转基因组 Transgenic	对照组 Control	转基因组 Transgenic	对照组 Control	转基因组 Transgenic	对照组 Control
母牛Cow	0	28.80	27.48	79.22	79.00	17.18	17.20	72.78	73.04
	3	125.80	115.40	92.22	91.11	24.82	22.88	110.95	110.44
	6	174.45	179.60	99.82	99.74	28.14	27.56	124.90	123.13
	9	218.60	225.15	106.23	106.56	30.45	31.06	136.50	134.70
	12	256.70	248.70	117.06	117.86	35.52	35.45	151.62	153.02
	15	271.20	275.60	122.52	121.79	36.04	36.22	157.53	155.44
	18	373.90	386.25	124.98	125.74	37.84	37.80	167.14	165.13
公牛Bull	0	30.60	28.70	80.00	79.70	17.50	17.40	73.40	73.20
	3	131.40	120.40	92.60	93.05	24.50	25.20	113.25	113.10
	6	201.35	192.20	102.40	102.00	29.20	28.10	126.30	125.45
	9	249.60	248.05	108.15	107.30	32.05	32.20	138.10	137.50
	12	297.10	292.50	118.20	117.80	36.10	35.85	154.00	154.20
	15	332.00	335.40	124.30	123.95	37.50	36.90	159.85	158.10
	18	443.70	456.35	127.20	126.20	39.60	39.50	168.60	168.25

性别 Sex	月龄/月 Month age/month	体质量 Body mass/kg		体高 Body height/cm		十字部宽 Height at hip cross/cm		胸围 Bust/cm
性别 Sex	月龄/月 Month age/month	转基因组 Transgenic	对照组 Control	转基因组 Transgenic	对照组 Control	转基因组 Transgenic	对照组 Control	转基因组 Transgenic	对照组 Control
母牛 Cow	0	33.10	32.41	73.90	72.47	20.90	21.33	76.21	77.53
	3	92.96	90.40	90.70	89.42	23.89	24.26	108.40	107.70
	6	154.10	152.50	99.90	99.50	28.02	28.33	125.90	124.80
	9	185.90	184.00	106.80	104.50	32.41	32.90	137.30	136.10
	12	231.20	229.30	114.70	113.20	35.66	34.45	150.40	147.20
	15	266.60	260.80	117.70	116.20	36.42	36.82	158.50	153.90
	18	310.90	299.80	120.20	119.20	37.92	37.04	169.20	165.70
公牛Bull	0	32.52	31.88	74.40	74.01	21.03	20.44	74.67	72.23
	3	99.70	100.90	93.18	92.69	26.78	26.34	118.30	116.50
	6	170.30	169.40	100.07	101.77	29.01	28.46	129.50	127.00
	9	220.70	218.60	108.40	107.30	32.03	31.62	153.70	147.80
	12	275.70	281.90	111.90	112.80	35.64	33.21	159.30	158.20
	15	344.50	332.80	115.90	115.70	37.02	37.45	163.20	161.30
	18	403.20	399.70	120.40	119.10	40.69	40.25	177.10	178.70