Recombinant nucleic acid fragment RecCR02BC16 and detection method thereof
A technology for recombining nucleic acid and fragments, which is applied in the field of recombinant nucleic acid fragments and its detection, can solve the problems of increased drug resistance of brown planthopper, increase of production cost, pollution of the environment, etc., and achieve the effect of improving the resistance of brown planthopper
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Embodiment 1
[0042] Example 1 Breeding of Recombinant Plants Introduced with Resistant Genome Fragment of Brown Planthopper
[0043] The materials used in this example are rice 'Shen 95B' and rice 'Hua 3418B'.
[0044] Rice 'Hua 3418B' has good resistance to brown planthopper, and it is speculated that it may be QBph3 (Hu et al., Molecular Breeding.2015,35:3) and Bph14 (Du et al., PNAS.2009,106(52):22163 on chromosome 3) -22168) located in the gene cluster region played a key role in the material's resistance to N. lugens planthopper.
[0045] During the breeding process of the recombinant plants, the molecular markers were used to perform prospect selection on the recombinant plants, and the adopted molecular markers for prospect selection were screened. Some of the molecular markers used were from the website http: / / www.gramene.org / , and some were designed by ourselves. The design method is to download the DNA sequence of the aforementioned segment referring to the MSU / TIGR annotati...
Embodiment 2
[0055] Example 2 Determination of Homologous Recombination Fragments After Introducing the Resistance Genome Fragments of Brown Planthopper
[0056] In order to determine the size of the imported N. lugens-resistant genome fragment, the homozygous single plant of the 'Shen 95B' imported fragment was sequenced for homologous recombination fragments on both sides of the target genome fragment. The N. lugens resistant genome recombination nucleic acid fragment contained in CR02BC16 was named RecCR02BC16.
[0057] It was preliminarily determined by the rice genome-wide breeding chip RICE60K detection results that RecCR02BC16 was located between two SNP markers F0335580364TC and R0335891133TC.
[0058] At the same time, three samples of ‘Shen 95B’, ‘Hua 3418B’ and CR02BC16 were sequenced using Miseq sequencing technology. The TruSeq Nano DNA LT Kit (illumina) kit was used for library establishment, the Library Quantification Kit–Universal (KAPA Biosystems) kit was used for quan...
Embodiment 3
[0068] Example 3 Resistance Identification of 'Shen 95B' After Introducing the Anti-BPH Genome Fragment
[0069] In order to identify the resistance effect, the new strain CR02BC16 bred by this application, the recipient parent 'Shen 95B', the donor parent 'Hua 3418B' (as a positive control), and the high-sensitivity brown planthopper variety 'Taizhong Zailai 1' (as a positive control) Negative control) for indoor resistance identification of brown planthopper, the identification method is as follows.
[0070] Soak the seeds and germination of each material in the room, and then sow them in plastic basins with grid lines. Each material is divided into 3 rows and a total of 45 plants are sown. When the two leaves are one heart, 10 plants are kept in each row, and a total of 30 plants grow healthy. Consistent plants are used for inoculation. The source of insects came from brown planthoppers collected from the field and bred indoors. Take 2-3 instar nymphs to the plants to ...
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