Creating a new germplasm of aneuploid Populus tomentosa with insect-resistance based on hybridization of transgenic 741 poplar and P. alba var. pyramidalis
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摘要:目的
以转BtCry3A基因三倍体741杨为母本与二倍体新疆杨为父本进行人工杂交，以期快速获得非整倍体毛白杨抗虫优良新种质。
方法采集转基因741杨雌花枝、新疆杨雄花枝进行人工授粉杂交，收集即将脱落的花序，通过胚挽救技术获得杂交子代。通过聚合酶链式反应（PCR）检测杂交子代是否含有BtCry3A基因；通过实时荧光定量PCR（RT-qPCR）对BtCry3A基因在母本与子代中的表达量进行分析。以二倍体新疆杨为参照，通过流式细胞技术检测杂交子代的倍性；并对子代幼苗表型进行初步观测分析。
结果（1）通过对未发育成熟种子进行胚挽救获得8个杂交子代，其中6个杂交子代后续生长状态良好，5个子代遗传了母本的BtCry3A基因。（2）RT-qPCR检测显示，BtCry3A基因在5个子代中的表达量均高于母本，其中3#表达量为母本12倍；（3）初步判定子代1#为超四倍体，子代2#、4#、8#为非整倍体，子代3#可能为非整倍体或四倍体；（4）杂交子代表型差异大，5个杂交子代叶形、叶片大小、节间距等均不相同，其中3#和8#生长势优于亲本。
结论通过转BtCry3A基因三倍体741杨与二倍体新疆杨杂交，快速获得了具有BtCry3A抗虫基因且发生形态变异的非整倍体毛白杨新种质，其中子代3#因抗虫基因表达量显著高于母本，且生长势优于双亲，可作为优良潜在非整倍体毛白杨新种质进行后续抗虫性测试。
Abstract:ObjectiveArtificial hybridization was carried out using transgenic triploid 741 poplar with BtCry3A as the female parent and diploid Populus alba var. pyramidalis as the male parent, in order to quickly obtain new germplasm of aneuploid Populus tomentosa with insect-resistance.
MethodCollecting female flower branches of transgenic 741 poplar and male flower branches of P. alba var. pyramidalis, hybrid progenies were obtained by embryo rescue technique from the inflorescence that was about to fall off. The seedlings with BtCry3A were identified by polymerase chain reaction, and further expression of BtCry3A was measured via real-time fluorescent quantitative polymerase chain reaction. Subsequently, conducted ploidy analysis of these hybrid progenies using flow cytometry method. Finally, preliminary phenotypic analysis of the offsprings was performed.
Result(1) Eight hybrid individuals were successfully generated by combing artificial hybridization and embryo rescue techniques, among which six hybrid offsprings showed good growth status. Five offsprings inherited the BtCry3A gene from the mother plants. (2) The BtCry3A gene exhibited enhanced expression levels in the offsprings compared with maternal parent, with individual 3# demonstrating a 12-fold increase. (3) 1# was hyper-tetraploid, 2#, 4# and 8# were aneuploid, while 3# may be aneuploid or tetraploid. (4) The hybrid offsprings exhibited diverse phenotype, the five hybrid individuals were different in leaf shape, leaf size and pitch spacing, with 3# and 8# showing superior growth vigor compared with parental lines.
ConclusionEmploying artificial hybridization between triploid 741 poplar with BtCry3A and diploid P. alba var. pyramidalis, we creat the novel germplasms of aneuploid P. tomentosa with BtCry3A and diverse phenotypic variation. Offspring 3# has significantly higher anti-insect gene expression than their mothers, and growth potential is better than parents. It can be used as an excellent potential aneuploid hair poplar new germplasm for subsequent insect resistance tests.
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图 1 花粉萌发（a）与胚挽救获得的杂交子代（b）
Figure 1. Pollen germination (a) and hybrid offsprings obtained from embryo rescuing (b)
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图 2 杂交子代中BtCry3A基因PCR（a）和RT-PCR（b）检测
a图中0为质粒，1为转基因741杨，2为新疆杨，3为野生型741杨，4 ~ 9为1#、2#、3#、4#、6#、8#子代。b图中1为新疆杨，2为野生型741杨，3为转基因741杨，4 ~ 9为1#、2#、3#、4#、6#、8#子代。 In figure a, 0 means plasmid, 1means transgenic 741 poplar, 2 means P. alba var. pyramidalis, 3 means wild type 741 poplar, 4−9 mean 1#, 2#, 3#, 4#, 6#, and 8# hybrids. In figure b, 1 means P. alba var. pyramidalis, 2 means wild type 741 poplar, 3 means transgenic 741 poplar, 4−9 mean 1#, 2#, 3#, 4#, 6#, and 8# hybrids.
Figure 2. Identification of BtCry3A in hybrid offsprings by PCR (a) and RT-PCR (b)
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图 3 不同株系BtCry3A基因RT-qPCR分析
**表示与野生型741杨相比，在0.01水平上存在极显著差异。** means extremly significant difference at 0.01 level compared with wild type 741 poplar.
Figure 3. Analysis of BtCry3A in different strains by RT-qPCR
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图 4 父母本与杂交子代核DNA流式细胞检测
a. 新疆杨；b. 转基因741杨；c ~ g. 杂交子代1#、2#、3#、4#、8#。a, P. alba var. pyramidalis; b, transgenic 741 poplar; c−g, 1#, 2#, 3#, 4#, 8# hybrid offsprings.
Figure 4. Detection of nuclear DNA of offspring and its parents by flow cytometer
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图 5 亲本与杂交子代幼苗表型前视图（a）和俯视图（b）
Figure 5. Phenotypes of parents and hybrid offspring front view (a) and vertical view(b)
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图 6 亲本与杂交子代幼苗表型差异分析
*表示与新疆杨相比在0.05水平上存在显著差异，**表示与新疆杨相比在0.01水平上存在极显著差异。下同。* means significant difference at 0.05 level compared with P. alba var. pyramidalis, ** means extremly significant difference at 0.01 level compared with P. alba var. pyramidalis. The same below.
Figure 6. Analysis of phenotypic difference between parents and hybrid offsprings
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图 7 亲本与杂交子代幼苗叶片表型差异分析
Figure 7. Analysis of leaf phenotypic difference between parent and hybrid offsprings
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图 8 亲本与杂交子代幼苗叶片纵切面
a. 新疆杨；b. 转基因741杨；c ~ g. 杂交子代1#/2#/3#/4#/8#。a, P. alba var. pyramidalis; b, transgenic 741 poplar; c−g, 1#, 2#, 3#, 4#, 8# hybrid offsprings.
Figure 8. Leaf longitudinal section of parents and hybrid offsprings
表 1 流式细胞分析法对杂交子代的倍性检测结果
Table 1 Detection results of nuclear ploidy of hybrid offsprings by flow cytometry method
样本
Sample
CV/%相对荧光强度
Relative fluorescence intensityF值
F value新疆杨 P. alba var. pyramidalis 6.96 ± 0.12 13 251.00 ± 21.23 2.00 741杨 741 poplar 6.07 ± 0.21 19 731.00 ± 129.25 2.98 1# 5.41 ± 0.28 28 297.33 ± 36.97 4.27 2# 5.86 ± 0.14 16 786.67 ± 129.60 2.53 3# 5.67 ± 0.19 26 006.67 ± 86.44 3.93 4# 6.56 ± 0.79 15 659.00 ± 259.90 2.36 8# 5.95 ± 0.22 24 763.00 ± 270.82 3.74 
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