Institute of Forest Biotechnology, Forestry College, Hebei Agricultural University, Baoding 071000, China.
Hebei Key Laboratory for Tree Genetic Resources and Forest Protection, Baoding 071000, China.
Int J Mol Sci. 2023 Aug 19;24(16):12952. doi: 10.3390/ijms241612952.
Multigene cotransformation has been widely used in the study of genetic improvement in crops and trees. However, little is known about the unintended effects and causes of multigene cotransformation in poplars. To gain insight into the unintended effects of T-DNA integration during multigene cotransformation in field stands, here, three lines (A1-A3) of × cv. Neva () carrying -- genes and three lines (B1-B3) of carrying -- genes were used as research objects, with non-transgenic as the control. Experimental stands were established at three common gardens in three locations and next generation sequencing (NGS) was used to identify the insertion sites of exogenous genes in six transgenic lines. We compared the growth data of the transgenic and control lines for four consecutive years. The results demonstrated that the tree height and diameter at breast height (DBH) of transgenic lines were significantly lower than those of the control, and the adaptability of transgenic lines in different locations varied significantly. The genotype and the experimental environment showed an interaction effect. A total of seven insertion sites were detected in the six transgenic lines, with B3 having a double-site insertion and the other lines having single copies. There are four insertion sites in the gene region and three insertion sites in the intergenic region. Analysis of the bases near the insertion sites showed that AT content was higher than the average chromosome content in four of the seven insertion sites within 1000 bp. Transcriptome analysis suggested that the differential expression of genes related to plant hormone transduction and lignin synthesis might be responsible for the slow development of plant height and DBH in transgenic lines. This study provides an integrated analysis of the unintended effects of transgenic poplar, which will benefit the safety assessment and reasonable application of genetically modified trees.
多基因共转化已广泛应用于作物和树木的遗传改良研究。然而，人们对杨树多基因共转化的非预期效应和原因知之甚少。为了深入了解田间林分多基因共转化过程中 T-DNA 整合的非预期效应，本研究以携带 -- 基因的三个品系（A1-A3）和携带 -- 基因的三个品系（B1-B3）为研究对象，以非转基因 为对照。在三个地点的三个普通花园中建立了实验林分，并使用下一代测序（NGS）来鉴定六个转基因品系中外源基因的插入位点。我们连续四年比较了转基因和对照品系的生长数据。结果表明，转基因品系的树高和胸径（DBH）显著低于对照，不同地点转基因品系的适应性差异显著。基因型和实验环境表现出互作效应。在六个转基因品系中检测到七个插入位点，B3 具有双位点插入，其他品系具有单拷贝插入。在基因区域有四个插入位点，在基因间区域有三个插入位点。插入位点附近碱基的分析表明，在七个插入位点中，有四个插入位点的 1000bp 内的 AT 含量高于平均染色体含量。转录组分析表明，与植物激素转导和木质素合成相关的基因的差异表达可能是导致转基因品系株高和 DBH 发育缓慢的原因。本研究对转基因杨树的非预期效应进行了综合分析，这将有利于转基因树木的安全性评估和合理应用。
