传统上，转基因的插入都是随机的，而且在育种中，转基因一般都先在可转化的实验室品系中完成，随后再渗入到当地优良的栽培品系中。但是在非遗传连锁的情况下，对于二倍体或者以二倍体形式遗传的多倍体作物来说，要通过这种方法获得n个独立转基因性状的纯合株系，几率为(1/4)n 。若育种者还需将x个非转基因性状导入同一品种，获得纯合子的几率为(1/4)n+x。例如，要获得3个转基因性状和7个优良性状的纯合株系，可能要筛选超过1,000,000个单株。所以当分离的转基因位点增多时，将大大增加育种难度。为保持单个转基因位点，一些研究人员会选择将新基因和已经导入基因组的基因在体外融合，构建新的载体并重新转化。但这意味着每增加一个新的性状，以前已经通过审定的性状就需要同新性状一起重新审定，耗费大量不必要的资源。另一选择是将新基因直接导入栽培品系，但因许多栽培品系转化困难使这项工作难以进行；再者，由于每一个独立转化获得的栽培品系被认为是独立的转化事件，需要单独审批，若要做多个栽培品系的产品，就需要在审批上花费大量时间和金钱。
针对上述问题，中科院华南植物园区永祥研究员等发明了可以自由使用（不涉及使用别人专利）的定点基因叠加系统，并在烟草中验证了该系统的可实现性（Hou et al., 2016. Molecular Plant 7:1756-1765）。考虑到要发挥该系统的真正价值，就需要应用于作物中，所以在农业部转基因重点专项（2010ZX08010-001）和中科院重点部署项目(KSZD-EW-Z-013)的支持下，区永祥研究员和韩志国副研究员首先在水稻上建立了使用该系统的所需要的起始目标系，并于2015年1月对相关成果进行了专利申请，最终在2018年7月获得了授权（发明专利号ZL 2015 1 0032720.0）。
该发明主要是关于能用于基因定点叠加的粳稻起始目标系的建立。包括构建了适用与基因叠加的载体：载体内含有用于基因叠加的目标位点：attP、attB序列，允许新的DNA分子通过重组酶Bxb1催化整合；获得整合了上述载体有效部分的并可用于水稻基因叠加的候选株系；确定了水稻染色体上适合整合新性状基因的7个基因组位点。在那些位点中，多个目的基因可在不影响其他基因的正常表达的情况下，能有效地叠加并高效地表达，这样一来，由于分离位点的数目得到减少，大大降低了将转基因从实验室品系导入农田品种过程中的工作量。
虽然该专利成果只限于目标系的建立，但在随后的试验中，区永祥研究员课题组的李如玉副研究员证明了该专利所确定的位点的确能对新基因进行有效叠加（未发表），切实地说明了这个专利的可应用性。所以此专利的获取将为水稻上的定点基因叠加/整合服务，进一步建立拥有我国有自主知识产权的定点基因转化技术平台奠定了基础。目前，区永祥课题组正在国家重点研发计划（2016YFD010904）中分别在大豆和棉花中建立相关体系。希望不久的将来能为相关科研和企业提供服务，使产学研能更好地结合。
The authorization of the invention patent of A Vector Suitable for Site Specific Gene Stacking from South China Botanical Garden
Conventionally, transgenic traits are introduced into transformable varieties before introgressing them out to field cultivars. However, For diploids and diploid-like allopolyploid plants, the ‘n’ number of unlinked transgenic loci can be assorted as homozygous into a single genome at a probability of (1/4)n. However, along with the ‘x’ number of other nontransgenic traits that breeders need to assemble into the same genome, the very low probability of (1/4)n+x makes line conversion difficult. For example, over 1,000,000 plants are needed for the co-assortment 7 elite traits plus 3 transgenic loci. To minimize the number of segregating transgenic loci, there are some available approaches. To minimize the number of segregating transgenic loci, the option of in vitro stacking prior to its introduction into the plant genome would mean the re-engineering and re-deregulating of previously introduced traits each time a new trait is introduced. The option of bypassing introgression by directly transforming field cultivars is also not practical as most field cultivars are difficult to transform. Moreover, each region-specific cultivar derived from individual transformation is regarded as an independent event and requires individual de-regulation. The available methods have a lot of limitations, therefore a more efficient method is required to speed up the development of transgenic cultivars.
Considering those obstacles for breeding and to maintain the number of segregating loci to a minimum, Dr. David Ow ’s group had established an open-source system in China, in planta recombinase-mediated gene stacking that can integrate a new DNA near the already existed transgenes (Hou et al., 2016 Molecular Plant 7:1756-1765). After that, the system was tried to apply in rice with the funding support from Chinese Ministry of Agriculture Grant (2010ZX08010-001) and The Key Project of Chinese Academy of Science (KSZD-EW-Z-013). Finally, Dr. David Ow and Dr. Zhi-Guo Han (a former member in Dr. OW’s lab) had established the target lines in Japonica rice for the gene stacking method. The related patent was filed in 2015 and granted in late 2018 (Patent No.，ZL 2015 1 0032720.0，Fig.1）.
The patent is mainly about establishing the target lines of Japonica rice with the target sites, consisting of either an attP or an attB sequence, to allow the integration of a new gene through Bxb1 integrase catalyzed site-specific recombination. This patent claims that 1) the vectors used for target line development 2) candidate target lines for later-on gene integrations 3) 7 target sites in rice genome that are suitable for new gene insertions. The effectiveness of these target lines for gene stacking was not described in the patent, but it has been confirmed by Dr. Ruyu Li in David Ow’s group, recently (unpublished). Within these target sites, the new genes can be correctly integrated, the insertion of new genes does not affect the expressions of the other genes in rice, the newly inserted genes have relatively high expression, etc.
The granted patent laid part of foundation for the future gene stacking service in rice. Currently, Dr. David Ow’s group are also applying the gene stacking system to soybean and cotton with the support of National Key Technologies R&D Program from Ministry of Science and Technology of the People’s Republic of China (2016YFD010904). Hopefully, this gene stacking system can be used in various crops and a great service platform with the intellectual property of our own country can be established for plant breeding.
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