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Clone and application of pleiotropic gene Ghd8 for controlling yield, florescence and plant height of rice grain

A technology for grain yield and plant height, applied in the field of plant genetic engineering, can solve the problems of delayed heading stage, reduced production and application value, etc., and achieves the effect of simple method and avoiding time-consuming

Inactive Publication Date: 2010-11-24
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This gene makes the plant height difference of the two allelic types of the near-isogenic line 30cm under long-day conditions, and the yiel

Method used

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  • Clone and application of pleiotropic gene Ghd8 for controlling yield, florescence and plant height of rice grain
  • Clone and application of pleiotropic gene Ghd8 for controlling yield, florescence and plant height of rice grain
  • Clone and application of pleiotropic gene Ghd8 for controlling yield, florescence and plant height of rice grain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Fine mapping of the Ghd8 gene

[0033] 1. Construction and evaluation of near-isogenic lines

[0034] according to figure 1 According to the technical route, the recombinant inbred line F7 population of ZS97 and HR5 was planted, and it was found that the phenotypes such as heading date, plant height, and grain number per ear appeared in the RIL39 family (Table 1). Select the N15 single plant of dwarf plant, early panicle and small panicle and the N16 single plant of tall plant, late panicle and large panicle, and refer to the rice genetic linkage map published by Temnykh et al. (Temnykh et al., 2000; Temnykh et al. ., 2001), selected 126 SSR marker pairs with polymorphism between N15 and N16 for background screening, and at the same time, F 2 The population was scanned for QTL (Table 2), F 2 The phenotype showed 3:1 segregation, plant height, ear size, co-segregation at anthesis and low-value traits were recessive (see attached figure 2 ). The QTL is lo...

Embodiment 2

[0044] Embodiment 2: Isolation and cloning of Ghd8 gene

[0045] According to the predicted candidate gene sequence, amplify the PCR fragment to screen the 9311BAC library, select the clone 50N15 containing the candidate gene LOC_Os08g07740, use the restriction endonuclease SacI to digest the positive clone, and perform subcloning to obtain a 10.26K b fragment. This fragment contains the sequence of 3.5kb upstream of the transcription initiation point and 5.7Kb downstream of the termination site, and only one complete gene of the candidate gene is contained in this fragment.

[0046] This fragment was ligated into the binary vector pCAMBIA1301 ( image 3 ), after the sequencing confirmed that the transformation vector was correct, the genetic transformation method mediated by Agrobacterium was used to obtain the transgenic rice. The specific steps of the transgene of the present invention are as follows:

[0047] The correctly cloned plasmid was introduced into the recessive...

Embodiment 3

[0241] Example 3: Searching for Ghd8 allelic types that differentiate different effects produced

[0242] Direct sequencing method with PCR products: 8 pairs of primers (see Image 6 ) to amplify each segment of the Ghd8 gene. The PCR product was digested by SAP and EXOI at 37°C for 1 hour (digestion reaction system 8 microliters), denatured at 80°C, and 2.5 microliters of the digested product was sequenced and amplified with the ABI sequencing kit Reaction (the first step is denaturation at 96°C for 2 minutes, the second step is denaturation at 96°C for 10 seconds, renaturation at 50°C, extension at 60°C for 4 minutes, this step is repeated for 33 cycles, the third step is 4°C for 10 minutes), the amplified product Precipitate with 95% ethanol: 3M sodium acetate (pH4.8) = 25:1, react with 26 microliters per well, collect the precipitate by centrifugation at 3000 g after precipitation for 15 minutes, take the supernatant and add 75 microliters of 70% ethanol After washing, ce...

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Abstract

The invention relates to the plant genetic engineering field, in particular to clone of a pleiotropic gene Ghd8 which affects yield, florescence and plant height of rice grain. In the invention, the gene Ghd8 is separated and cloned by experiments such as establishment of a near-isogenic line of a target gene, preliminary location, comparative sequencing, genetic transformation and complementation and the like, wherein the sequence of the gene Ghd8 is shown as SEQ ID NO:2. Compared with a recessive allelic single plant, the yield of a dominant allelic single plant is increased by 58%, the plant height thereof is increased by 25.6%, but the heading stage thereof is only increased by 9 days. By means of results of Ghd8 main mutation site and trait correlation analysis and haploid cluster analysis of protein sequences of different varieties, the near-isogenic lines of different allelic types are established to obtain four favorable Ghd8 allelic types, wherein, the Ghd8-9311 and Ghd8-ruf allelic types can increase yield of the rice grain but not delay blossoming, thus being applicable to breeding the rice grain in areas with good light and temperature conditions; and Ghd8-MH63 and Ghd8-Nip allelic types are not photosensitive, thus being applicable to increasing yield and breeding the rice grain in areas under a short-day condition.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering. It specifically relates to the cloning and application of a pleiotropic gene Ghd8 affecting rice grain yield, flowering period and plant height. The present invention fine-positions the quantitative trait locus gene with huge benefits, and uses the genetic transformation mediated by Agrobacterium to verify the function of the gene; The function of different allelic types of the gene was verified. Background technique [0002] Currently, the world's population is approaching 7 billion, half of which depend on rice as their staple food. To meet the growing population's demand for food, it is of great significance to increase the yield of rice per unit area, especially under the condition that the area of ​​arable land is decreasing in today's economic development. [0003] Rice yield is a complex agronomic trait influenced by many factors, and its constituent factors mainly inc...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415A01H5/00
Inventor 邢永忠鄢文豪余四斌王鹏
Owner HUAZHONG AGRI UNIV
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