转基因白桦杂交子代种子活力及外源基因遗传规律分析

doi:10.7525/j.issn.1673-5102.2021.04.012

摘要/Abstract

摘要：

探索目的基因在转基因白桦杂交子代群体中的传递规律，为后续进一步跟踪调查，实现转基因白桦的聚合育种提供帮助。以1年生BpAP1、TabZIP转基因白桦杂交T1代和5年生BpGH3.5、BpCCR转基因白桦为研究对象，测定杂交子代种子千粒质量、发芽率和发芽势等活力指标，同时采用PCR扩增法检测目的基因，调查杂交T2代白桦苗高。杂交子代各家系间种子千粒质量、种子活力指标和苗高等在0.01水平上差异显著，各性状的家系遗传力均高于85.00%。研究结果表明，目的基因在T2代群体中的传递规律不符合孟德尔遗传规律，雌、雄配子目的基因的传递效率分别为30.00%、50.00%左右，同时未获得3目的基因聚合的杂交T2代个体。目的基因的随机插入降低了白桦杂交T2代种子活力，未获得3基因聚合的白桦杂交子代，目的基因的聚合对杂交子代个体的生长发育产生了不利影响，甚至产生致死现象。

关键词: 转基因白桦, 种子活力, 聚合育种, 目的基因检测, 传递规律

Abstract:

To explore the transmission mode of target gene in hybrid offspring of transgenic birch for further follow-up investigation， it is helpful to realize polymerization breeding for study on transgenic birch. In this study， the growth traits including thousand-seed weight，germination rate，germination potential and vigor index for offspring of transgenic birch were measured respectively. The offspring included families of T1 cross generation of 1-year-old BpAP1 and TabZIP transgenic birch， 5-year-old BpGH3.5 and BpCCR transgenic birch and half-sibling families of open pollination. The results showed that thousand-seed weight， seed vigor index and seedling height were all significantly different at 0.01 level among those families respectively， and family heritability of each trait was higher than 85.00%. Results showed that the transmission mode of target gene in T2 generation population does not accord with mendelian inheritance. The transfer efficiency of target genes of female and male gametes was about 30.00% and 50.00% respectively， however T2 hybrid polymerized with the 3 target genes was unattained. The random insertion of target gene reduced the seed vigor of T2 generation hybrid. We did not obtain birch hybrids with the 3 target genes. The polymerization of the target gene had an adverse effect on the growth and development of the hybrid offspring， and even death.

Key words: transgenic birch, seed vigor, pyramiding breeding, detection of target gene, inherited rules

中图分类号:

S792.153

吕东林, 李腾, 郭译文, 姜静, 黄海娇. 转基因白桦杂交子代种子活力及外源基因遗传规律分析[J]. 植物研究, 2021, 41(4): 564-572.

Dong-Lin LÜ, Teng LI, Yi-Wen GUO, Jing JIANG, Hai-Jiao HUANG. Determination of Seed Vigor and Genetic Analysis of Foreign Genes in Different Transgenic Birch Hybrids[J]. Bulletin of Botanical Research, 2021, 41(4): 564-572.

图/表 9

表1

表2

图1

表3

表4

不同杂交子代家系种子千粒质量、发芽率、发芽势、发芽指数、平均发芽时间的多重比较

序号 Number	家系 Family	千粒质量 Thousand-seed weight(mg)	家系 Family	发芽率 Germination rate(%)	家系 Family	发芽势 Germination potential(%)	家系 Family	发芽指数 Germination index	家系 Family	平均发芽时间 Mean time of germination(d)
1	AG20-6	381.50±30.45a	AG20-6	54.02±7.47a	AG20-4	10.95±3.87abc	CA13	16.14±2.06a	AGB5-2	6.66±0.31a
2	AG20-4	279.63±12.55b	CA13	46.09±4.22b	AG20-5	9.40±3.32g	AG20-6	15.26±1.84a	AG20-4	5.81±0.89b
3	AG20-8	264.50±11.84c	AG20-8	45.96±3.19b	AG20-7	8.12±2.87cde	AG13-1	14.20±2.30ab	AG20-5	5.58±0.82bc
4	AG13-1	262.00±27.00c	AG20-7	45.94±8.12b	AG20-6	7.47±3.74a	AG20-7	12.35±3.30bc	GA22	5.58±0.66bc
5	AG13-4	240.50±7.78d	AG20-3	42.47±3.31bc	AG13-2	4.72±1.67def	AG20-3	12.20±3.30bc	GA8	5.47±0.66bcd
6	ACB1-1	231.50±10.03de	AG13-1	42.25±4.30bc	AG13-1	4.30±1.52ab	AG20-8	11.29±4.11cd	AG13-3	5.41±0.57bcd
7	CA13	230.38±16.26de	AG20-4	40.23±10.95bcd	CA13	4.22±1.49a	AG20-1	10.92±2.47cde	AG13-4	5.36±0.13bcd
8	G22	220.75±11.73e	AG20-1	38.96±4.09cd	AG13-3	4.20±1.48defg	G8	9.96±1.11cdef	AG20-2	5.27±0.69bcde
9	AG20-1	219.63±15.99e	AGB1-1	38.62±3.55cd	AG20-1	4.09±1.45def	AG13-5	9.87±2.46cdef	G22	5.25±0.46bcde
10	GA22	181.88±5.74f	G22	36.66±3.70cde	AG13-5	3.97±1.4fg	AGB1-1	9.26±1.03def	ACB2-1	5.03±0.61cde
11	AGB5-1	179.25±14.97fg	ACB1-1	35.76±3.34def	GA8	3.76±1.33fg	ACB1-1	8.94±2.05def	AGB5-3	4.94±1.07cdef
12	AG20-2	173.88±9.93fgh	G8	35.19±1.88defg	G22	3.70±1.31def	C13	8.79±1.53def	AG13-2	4.82±0.74cdefg
13	AG20-5	171.13±6.98fgh	AG13-2	35.03±4.72defg	AGB5-2	3.67±2.12h	AG20-4	8.29±4.704ef	AC15-1	4.79±0.66defgh
14	AC15-2	170.71±8.56fgh	C13	34.95±2.55defg	AGB1-1	3.55±2.05def	AGB5-1	7.64±1.03fg	AC15-2	4.77±0.96defgh
15	GA8	170.50±9.37fgh	AG13-3	32.14±4.20efgh	AC15-3	3.43±1.21h	AG13-2	7.48±2.85fg	AGB1-1	4.56±0.23efghi
16	G8	169.88±6.45fgh	AGB5-1	31.98±2.56efgh	ACB1-1	3.34±1.18efg	G22	7.26±1.50fg	AG20-6	4.55±0.33efghi
17	C13	169.13±8.01fgh	AG13-4	30.00±0.94fgh	AG20-3	3.31±1.17bcd	AG13-3	5.53±1.97gh	AG20-7	4.52±0.30efghi
18	AG20-7	168.13±9.75fgh	AG13-5	29.69±3.97ghi	AG20-8	3.19±1.3def	AG13-4	4.90±0.44gh	AG20-8	4.24±0.10fghij
19	AGB1-1	163.00±15.72gh	GA22	28.84±2.77hi	AG20-2	3.13±1.11g	GA8	4.19±1.53hi	ACB1-1	4.16±0.49ghij
20	AC15-3	158.38±8.31h	GA8	26.94±3.76hij	AGB5-3	2.85±1.01h	GA22	4.05±1.01hi	C13	4.05±0.46hijk
21	AG13-3	141.88±7.85i	AG20-5	24.20±9.40ij	GA22	2.77±0.98fg	AG20-5	3.90±3.38hi	AG20-3	3.99±0.57ijkl
22	AG13-2	138.50±9.10i	AG20-2	22.95±3.13j	AGB5-1	2.56±0.91efg	AG20-2	3.18±1.21hij	AGB5-1	3.92±0.51ijkl
23	AG20-3	137.38±6.67i	AC15-1	15.41±1.66k	C13	2.55±0.90efg	AC15-3	1.71±0.94ij	AG20-1	3.92±0.43ijkl
24	AC15-1	135.13±7.02i	AC15-3	13.05±3.43kl	AC15-2	2.39±0.90h	AC15-1	1.60±0.44ij	G8	3.58±0.16jkl
25	ACB2-1	128.50±22.98i	AGB5-2	12.13±3.67kl	ACB2-1	2.24±0.79h	ACB2-1	0.84±0.31j	CA13	3.33±0.14klm
26	AGB5-2	108.00±4.00j	ACB2-1	11.35±2.24kl	G8	1.88±0.66def	AGB5-2	0.73±0.41j	AC15-3	3.30±0.34lm
27	AG13-5	102.50±13.35jk	AGB5-3	9.84±2.85kl	AC15-1	1.66±0.59h	AGB5-3	0.67±0.42j	AG13-1	3.27±0.1lm
28	AGB5-3	90.38±4.81k	AC15-2	8.13±2.39l	AG13-4	0.94±0.66g	AC15-2	0.48±0.34j	AG13-5	2.77±0.21m

表4

表5

表6

表7

表8

参考文献 26

1	李付广，袁有禄.棉花分子育种进展与展望［J］.中国农业科技导报，2011，13（5）：1-8.
	Li F G，Yuan Y L.Progress and prospect of cotton molecular breeding［J］.Journal of Agricultural Science and Technology，2011，13（5）：1-8.
2	Hittalmani S，Parco A，Mew T V，et al.Fine mapping and DNA marker-assisted pyramiding of the three major genes for blast resistance in rice［J］.Theoretical and Applied Genetics，2000，100（7）：1121-1128.
3	Shah B H，Ding X H，Zeng L X，et al.Pyramiding four bacterial blight resistance genes into rice cultivars in south China［J］.Molecular Plant Breeding，2006，4（4）：493-499.
4	董娜，张亚娟，张军刚，等.分子标记辅助小麦抗白粉病基因Pm21和Pm13聚合育种［J］.麦类作物学报，2014，34（12）：1639-1644.
	Dong N，Zhang Y J，Zhang J G，et al.Molecular marker assisted pyramid breeding of powdery mildew resistance gene Pm21 and Pm13［J］.Journal of Triticeae Crops，2014，34（12）：1639-1644.
5	Matsumoto Y，Watanabe N，Kuboyama T.Cross-species transferability of 86 cucumber（Cucumis sativus L.）microsatellite markers to gherkin（C.anguria L.）［J］.Scientia Horticulturae，2012，136：110-114.
6	朱明涛，孙亚林，郑莎，等.分子标记辅助聚合番茄抗病基因育种［J］.园艺学报，2010，37（9）：1416-1422.
	Zhu M T，Sun Y L，Zheng S，et al.Pyramiding disease resistance genes by molecular marker-assisted selection in tomato［J］.Acta Horticulturae Sinica，2010，37（9）：1416-1422.
7	Ritonga F N，Chen S.Physiological and molecular mechanism involved in cold stress tolerance in plants［J］.Plants，2020，9（5）：560.
8	Chen S，Lin X，Zhang D W，et al.Genome-wide analysis of NAC gene family in Betula pendula［J］.Forests，2019，10（9）：741.
9	李天芳，姜静，杨传平，等.我国白桦育种研究概况［J］.江苏林业科技，2008，35（2）：47-49.
	Li T F，Jiang J，Yang C P，et al.Status of breeding research on Betula platyphylla in China［J］.Journal of Jiangsu Forestry Science & Technology，2008，35（2）：47-49.
10	Huang H J，Wang S，Jiang J，et al.Overexpression of BpAP1 induces early flowering and produces dwarfism in Betulaplatyphylla × Betulapendula［J］.Physiologia Plantarum，2014，151（4）：495-506.
11	李园园，杨光，韦睿，等.转TabZIP基因白桦的获得及耐盐性分析［J］.南京林业大学学报：自然科学版，2013，37（5）：6-12.
	Li Y Y，Yang G，Wei R，et al.TabZIP transferred Betulaplatyphylla generation and salt tolerance analysis［J］.Journal of Nanjing Forestry University：Natural Science Edition，2013，37（5）：6-12.
12	Zhang W B，Wei R，Chen S，et al.Functional characterization of CCR in birch（Betulaplatyphylla × Betulapendula）through overexpression and suppression analysis［J］.Physiologia Plantarum，2015，154（2）：283-296.
13	Yang G，Chen S，Jiang J.Transcriptome analysis reveals the role of BpGH3.5 in root elongation of Betulaplatyphylla × B. pendula［J］.Plant Cell，Tissue and Organ Culture，2015，121（3）：605-617.
14	孟德恺，徐志鹏，刘宁宁，等.白桦转BpGH3.5基因叶片早衰突变株的光合特性及生长分析［J］.南京林业大学学报：自然科学版，2019，43（5）：37-43.
	Meng D K，Xu Z P，Liu N N，et al.Characterization of photosynthetic and growth traits of precocious leaf senescence mutant of BpGH3.5 transgenic lines in Betula platyphylla［J］.Journal of Nanjing Forestry University：Natural Science Edition，2019，43（5）：37-43.
15	张宇，陈肃，高源，等.BpMYB4基因在白桦中的遗传转化及低温胁迫应答反应［J］.南京林业大学学报：自然科学版，2019，43（1）：25-31.
	Zhang Y，Chen S，Gao Y，et al.Functional study of BpMYB4 in birch response to low temperature stress［J］.Journal of Nanjing Forestry University：Natural Science Edition，2019，43（1）：25-31.
16	任丽，董京祥，杨洋，等.白桦BpTCP7基因启动子的克隆及表达分析［J］.南京林业大学学报：自然科学版，2019，43（1）：32-38.
	Ren L，Dong J X，Yang Y，et al.Cloning and expression analysis of BpTCP7 promoter from Betula platyphylla［J］.Journal of Nanjing Forestry University：Natural Science Edition，2019，43（1）：32-38.
17	安琳君，栾嘉豫，任丽，等.白桦BpTCP8基因生物信息学及表达特性分析［J］.南京林业大学学报：自然科学版，2019，43（5）：67-73.
	An L J，Luan J Y，Ren L，et al.Bioinformatics and expression characteristics analysis of BpTCP8 in Betulaplatyphylla Suk.［J］.Journal of Nanjing Forestry University：Natural Science Edition，2019，43（5）：67-73.
18	陈素素，张嫚嫚，于洪淼，等.转基因白桦不同杂交组合的种子活力测定及外源基因的遗传规律分析［J］.北京林业大学学报，2016，38（1）：36-42.
	Chen S S，Zhang M M，Yu H M，et al.Determination of seed vigor and genetic analysis of foreign genes in different transgenic birch hybrids［J］.Journal of Beijing Forestry University，2016，38（1）：36-42.
19	赵洁，杜沙沙，邱彤，等.转双Bt基因巨霸杨外源基因表达及抗虫性检测［J］.东北林业大学学报，2016，44（3）：47-51.
	Zhao J，Du S S，Qiu T，et al.Exogenous gene expression and insect resistance detection of transgenic Populusdeltocdes ‘55/56’ × P. deltocdescv ‘2KEN8’ with double Bt gene［J］.Journal of Northeast Forestry University，2016，44（3）：47-51.
20	胥猛，潘惠新，张博，等.林木遗传改良中的分子生物学研究进展［J］.林业科学，2009，45（1）：136-143.
	Xu M，Pan H X，Zhang B，et al.Molecular biology applied in the improvement process of forest trees［J］.Scientia Silvae Sinicae，2009，45（1）：136-143.
21	张启军，吕川根，虞德容，等.水稻抗病基因聚合育种研究进展［J］.中国农学通报，2008，24（8）：57-62.
	Zhang Q J，Lv C G，Yu D R，et al.Progress on the research of pyramiding resistant genes in rice［J］.Chinese Agricultural Science Bulletin，2008，24（8）：57-62.
22	王朔，黄海娇，杨光，等.转基因白桦杂种T₁代的生长发育及AP1基因的遗传分析［J］.北京林业大学学报，2016，38（9）：1-7.
	Wang S，Huang H J，Yang G，et al.Growth and developmental analysis of T₁ generation from BpAP1 transgenic birch［J］.Journal of Beijing forestry university，2016，38（9）：1-7.
23	周静，弭晓菊，崔继哲.植物转基因的非孟德尔遗传［J］.分子植物育种，2006，4（5）：614-620.
	Zhou J，Mi X J，Cui J Z.Non-mendelian inheritance of transgenes in plants［J］.Molecular Plant Breeding，2006，4（5）：614-620.
24	Peña L，Martín-Trillo M，Juárez J，et al.Constitutive expression of ArabidopsisLEAFY or APETALA1 genes in citrus reduces their generation time［J］.Nature Biotechnology，2001，19（3）：263-267.
25	Kotoda N，Wada M，Kusaba S，et al.Overexpression of MdMADS5，an APETALA1-like gene of apple，causes early flowering in transgenic Arabidopsis［J］.Plant Science，2002，162（5）：679-687.
26	Yin Z，Plader W，Malepszy S.Transgene inheritance in plants［J］.Journal of Applied Genetics，2004，45（2）：127-144.

杂交母本 Female parent	杂交父本 Male parent	组合代码 Group code	子代代码 Offspring code
BpAP1转基因白桦T₁代 T₁ generation of BpAP1 transgenic birch	BpGH3.5转基因白桦T₀代 T₀ generation of BpGH3.5 transgenic birch	P13-1×G7	AG13-1
		P13-2×G7	AG13-2
		P13-3×G7	AG13-3
		P13-4×G7	AG13-4
		P13-5×G7	AG13-5
		P20-1×G8	AG20-1
		P20-2×G8	AG20-2
		P20-3×G8	AG20-3
		P20-4×G8	AG20-4
		P20-5×G8	AG20-5
		P20-6×G8	AG20-6
		P20-7×G8	AG20-7
		P20-8×G8	AG20-8
	BpCCR转基因白桦T₀代 T₀ generation of BpCCR transgenic birch	P15-1×C13	AC15-1
		P15-2×C13	AC15-2
		P15-3 ×C13	AC15-3
BpAP1TabZIP转基因白桦T₁代 T₁ generation of BpAP1 and TabZIP transgenic birch	BpCCR转基因白桦T₀代 T₀ generation of BpCCR transgenic birch	PB1-2×C13	ACB1-1
	BpCCR转基因白桦T₀代 T₀ generation of BpCCR transgenic birch	PB2-1×C13	ACB2-1
	BpGH3.5转基因白桦T₀代 T₀ generation of BpGH3.5 transgenic birch	PB1-1×G7	AGB1-1
		PB5-1×G7	AGB5-1
		PB5-2×G22	AGB5-2
		PB5-3×G22	AGB5-3
BpGH3.5转基因白桦T₀代 T₀ generation of BpGH3.5 transgenic birch	BpAP1转基因白桦T₁代 T₁ generation of BpAP1 transgenic birch	G8×AP1	GA8
		G22×AP1	GA22
BpCCR转基因白桦T₀代 T₀ generation of BpCCR transgenic birch		C13×AP1	CA13
BpGH3.5转基因白桦T₀代(G8) T₀ generation of BpGH3.5 transgenic birch(G8)	自由授粉 Open pollination	——	G8
BpGH3.5转基因白桦T₀代(G22) T₀ generation of BpGH3.5 transgenic birch(G22)			G22
BpCCR转基因白桦T₀代(C13) T₀ generation of BpCCR transgenic birch(C13)			C13

性状 Trait	均值 Average	自由度 df	均方MS Mean square	F值 F value	标准差 Standard deviation	变幅 Variable amplitude	变异系数 Coefficient of variation(%)	家系遗传力 Heritability
千粒质量 Thousand-seed weight(mg)	181.25	27	2 189.728	134.963^**	57.16	84.00~410.00	31.54	0.992 6
发芽率 Germination rate(%)	30.94	27	1 024.502	47.043^**	12.70	5.74~64.90	41.05	0.978 7
发芽势 Germination potential(%)	19.76	27	881.696	19.369^**	12.72	1.00~57.00	64.37	0.948 4
发芽指数 Germination index	7.24	27	148.012	30.337^**	4.98	0.17~19.09	68.78	0.967 0
平均发芽时间 Mean time of germination(d)	4.53	27	5.441	16.289^**	1.02	2.33~7.00	22.52	0.938 6

性状 Trait	千粒质量 Thousand-seed weight（mg）	发芽率 Germination rate(%)	发芽势 Germination potential(%)	发芽指数 Germination index
千粒质量 Thousand-seed weight(mg)	1	0.611^**	0.603^**	0.528^**
发芽率 Germination rate(%)		1	0.858^**	0.915^**
发芽势 Germination potential(%)			1	0.820^**
发芽指数 Germination index				1

双亲 Parents		子代 Offspring	检测株数 Detection of the number	PCR检测阳性的个体数（株）及遗传分离理论比的卡方检测 Positive individual number of PCR and genetic separation ratio of Chi-square testing
♀	♂	子代 Offspring	检测株数 Detection of the number	BpGH3.5	BpAP1	BpGH3.5+BpAP1	均无 None	预测遗传分离比例 Genetic separation ratio	Χ²	Χ²_（0.05）n	插入位点数 Insertion sites
P20-1	G8	AG20-1	24	7	0	5	12	1∶1∶1∶1	12.33	7.82	不能确定 Uncertain
PB5-1	G7	AGB5-1	31	5	6	7	13	1∶1∶1∶1	5.00		1
P20-4	G8	A14	26	10	6	6	4	1∶1∶1∶1	2.92		1
双亲 Parents		子代 Offspring	检测株数 Detection of the number	PCR检测阳性的个体数（株）及遗传分离理论比的卡方检测 Positive individual number of PCR and genetic separation ratio of Chi-square testing
♀	♂	子代 Offspring	检测株数 Detection of the number	TabZIP	BpAP1	TabZIP+ BpAP1	均无 None	预测遗传分离比例 Genetic separation ratio	Χ²	Χ²_（0.05）n	插入位点数 Insertion sites
PB2-1	C13	A30	12	1	2	3	6	1∶1∶1∶1	4.67	7.82	1

[1]	杨建伟, 李宗艳, 冯尧, 任书娴, 胡梦露, 叶松菩. 束花石斛的繁育生物学特性[J]. 植物研究, 2023, 43(1): 150-160.
[2]	林琳, 高宇婷, 程福山, 辛本花, 王贵春, 夏富才, 穆怀志. 赛黑桦不同半同胞家系种子活力比较[J]. 植物研究, 2020, 40(1): 125-132.
[3]	孙丰坤, 李思达, 李吉祥, 陈晓慧, 曾凡锁. 外源NO对转基因白桦外源基因表达及DNA甲基化的影响[J]. 植物研究, 2017, 37(6): 870-875.
[4]	王广印;陈碧华;陈　群;莫一帆;高爱霞. 萝卜种子萌发的逆温耐性诱导研究[J]. 植物研究, 2008, 28(4): 482-485.