Breeding method for regulating mir156 and its target gene ipa1 to simultaneously improve rice disease resistance and yield

Abstract

The invention provides the application of a gene capable of improving rice agricultural production traits and bacterial blight resistance, including rice miR156 and the application of a target gene of rice miR156 in improving rice resistance to bacterial blight. It also provides a method for cultivating a transgenic rice resistant to bacterial blight, the method comprising constructing an overexpression vector containing OsSPL7 or OsSPL14, transforming the constructed overexpression vector into rice to up-regulate the expression of rice OsSPL7 or IPA1 / OsSPL14, and obtain an anti-white leaf blight blight transgenic rice; or down-regulate the expression of miR156 in rice crops, up-regulate the expression of miR156 target genes OsSPL7 and OsSPL14 (IPA1), and obtain bacterial blight-resistant transgenic rice. Based on the premise of not affecting the growth and development and even improving the agronomic traits such as rice plant type and panicle type, the present invention uses microRNA as a starting point to provide a method that can not only affect rice plant type and panicle type, but also enhance rice bacterial blight resistance genes. resource.

Description

technical field

[0001] The invention belongs to the field of rice genetics and breeding, and specifically relates to the breeding application of miR156 and its target genes OsSPL7 and IPA1 (that is, OsSPL4) to improve yield and enhance disease resistance. Rice miR156 negatively regulates the expression of SPL (Squamosapromoter binding protein like) family genes, and down-regulating the expression of miR156 will increase the expression of target genes OsSPL7 and OsSPL14 (IPA1). Using rice genetic transformation technology, miR156 target gene mimic technology (MIMIC) was used to inhibit the function of miR156 and overexpress two target genes of miR156, OsSPL7 and OsSPL14 (IPA1), to obtain stable transgenic rice. The results showed that down-regulation of miR156 and up-regulation of OsSPLs could enhance bacterial blight resistance and yield in rice. Background technique

[0002] microRNA (miRNA) is a kind of non-coding RNA widely present in plants and animals, and its length i...

Examples

Embodiment 1

[0063] Example 1 miR156northern analysis and OsSPLs expression pattern identification

[0064] The leaves of rice Nipponbare in the tillering stage were injected with Xanthobacterium blight PXO99A (OD value 0.6), and the inoculated leaves were sampled at 7 time points (0, 6, 12, 24, 48, 72, 120 hpi). The samples were mixed with three leaves, and the total RNA of 7 inoculation periods was extracted to identify the expression of miR156 and the target gene OsSPLs gene. (hpi: hours postinoculation, hours after inoculation)

[0065] 1.1 Culture of bacterial blight PXO99A:

[0066] PSA medium

[0067]

[0068] 1.2 Trizol method to extract total RNA:

[0069] The rice leaf samples taken during the 7 inoculation periods were respectively operated as follows to obtain the total RNA of the 7 inoculation periods:

[0070] (1) Weigh 0.1 g of fresh leaves, grind them quickly in liquid nitrogen with a mortar, and transfer them to a 1.5 mL centrifuge tube (pre-cooled with liquid nitro...

Embodiment 2

[0104] Example 2 Construction of rice miR156-specifically down-regulated transgenic plants using miRNA target gene simulation technology

[0105] The rice used for transformation is: wild type rice Zhonghua 11 (ZH11).

[0106] 2.1 The construction method of the overexpression vector MIM156OE containing miR156MIMIC is as follows:

[0107] 1) Use IPSF and IPSR primers to clone the cDNA sequence of the Arabidopsis gene IPS, digest with BamHI and SacI, and connect the fragment to the PBSk vector.

[0108] 2) Using the pairing of MIM156-I primers and IPSR primers and MIM156R primers and IPSF primers, using the plasmid after IPS ligated with PBSK as a template, two sequences containing terminal homology were amplified.

[0109] 3) Using the two sequences obtained in the second step as templates, using overlapping PCR technology and using IPSF and IPSR as primers, amplify the IPS fragment (IPS-MIMI156) containing the sequence of MIMIC156.

[0110] 4) The target fragment was amplifi...

Embodiment 3

[0119] Example 3 Rice mutants up-regulated by miR156

[0120] The rice used for transformation is: wild type rice Zhonghua 11 (ZH11).

[0121] 3.1 The construction method of the overexpression vector miR156fOE containing miR156f is as follows:

[0122] Using rice Nipponbare DNA as a template, using miR156fOE-F and miR156fOE-R primers to clone rice miR156 precursor miR156f, and connecting the p1301-35SNos plasmid to obtain the overexpression vector mi156fOE containing miR156f (shown in SEQ ID NO.2).

[0123] miR156fOE-F: GGGATCCttttgggtggtggcagttga (SEQ ID NO. 31)

[0124] miR156fOE-R: GGGTACcaaagccgtctcctccctcc (SEQ ID NO. 32)

[0125] 3.2 Transformation and infection methods are the same as in Example 2.

[0126] The miR156fOE vector was transformed into commercially available Agrobacterium EHA105 by freeze-thaw method, and then genetically transformed into wild-type rice Zhonghua 11 (ZH11). By overexpressing miR156f, the precursor of miR156, two transgenic lines miR156fOE...

technical field

[0001] The invention belongs to the field of rice genetics and breeding, and specifically relates to the breeding application of miR156 and its target genes OsSPL7 and IPA1 (that is, OsSPL4) to improve yield and enhance disease resistance. Rice miR156 negatively regulates the expression of SPL (Squamosapromoter binding protein like) family genes, and down-regulating the expression of miR156 will increase the expression of target genes OsSPL7 and OsSPL14 (IPA1). Using rice genetic transformation technology, miR156 target gene mimic technology (MIMIC) was used to inhibit the function of miR156 and overexpress two target genes of miR156, OsSPL7 and OsSPL14 (IPA1), to obtain stable transgenic rice. The results showed that down-regulation of miR156 and up-regulation of OsSPLs could enhance bacterial blight resistance and yield in rice. Background technique

[0002] microRNA (miRNA) is a kind of non-coding RNA widely present in plants and animals, and its length i...

Examples

Embodiment 1

[0063] Example 1 miR156northern analysis and OsSPLs expression pattern identification

[0064] The leaves of rice Nipponbare in the tillering stage were injected with Xanthobacterium blight PXO99A (OD value 0.6), and the inoculated leaves were sampled at 7 time points (0, 6, 12, 24, 48, 72, 120 hpi). The samples were mixed with three leaves, and the total RNA of 7 inoculation periods was extracted to identify the expression of miR156 and the target gene OsSPLs gene. (hpi: hours postinoculation, hours after inoculation)

[0065] 1.1 Culture of bacterial blight PXO99A:

[0066] PSA medium

[0067]

[0068] 1.2 Trizol method to extract total RNA:

[0069] The rice leaf samples taken during the 7 inoculation periods were respectively operated as follows to obtain the total RNA of the 7 inoculation periods:

[0070] (1) Weigh 0.1 g of fresh leaves, grind them quickly in liquid nitrogen with a mortar, and transfer them to a 1.5 mL centrifuge tube (pre-cooled with liquid nitro...

Embodiment 2

[0104] Example 2 Construction of rice miR156-specifically down-regulated transgenic plants using miRNA target gene simulation technology

[0105] The rice used for transformation is: wild type rice Zhonghua 11 (ZH11).

[0106] 2.1 The construction method of the overexpression vector MIM156OE containing miR156MIMIC is as follows:

[0107] 1) Use IPSF and IPSR primers to clone the cDNA sequence of the Arabidopsis gene IPS, digest with BamHI and SacI, and connect the fragment to the PBSk vector.

[0108] 2) Using the pairing of MIM156-I primers and IPSR primers and MIM156R primers and IPSF primers, using the plasmid after IPS ligated with PBSK as a template, two sequences containing terminal homology were amplified.

[0109] 3) Using the two sequences obtained in the second step as templates, using overlapping PCR technology and using IPSF and IPSR as primers, amplify the IPS fragment (IPS-MIMI156) containing the sequence of MIMIC156.

[0110] 4) The target fragment was amplifi...

Embodiment 3

[0119] Example 3 Rice mutants up-regulated by miR156

[0120] The rice used for transformation is: wild type rice Zhonghua 11 (ZH11).

[0121] 3.1 The construction method of the overexpression vector miR156fOE containing miR156f is as follows:

[0122] Using rice Nipponbare DNA as a template, using miR156fOE-F and miR156fOE-R primers to clone rice miR156 precursor miR156f, and connecting the p1301-35SNos plasmid to obtain the overexpression vector mi156fOE containing miR156f (shown in SEQ ID NO.2).

[0123] miR156fOE-F: GGGATCCttttgggtggtggcagttga (SEQ ID NO. 31)

[0124] miR156fOE-R: GGGTACcaaagccgtctcctccctcc (SEQ ID NO. 32)

[0125] 3.2 Transformation and infection methods are the same as in Example 2.

[0126] The miR156fOE vector was transformed into commercially available Agrobacterium EHA105 by freeze-thaw method, and then genetically transformed into wild-type rice Zhonghua 11 (ZH11). By overexpressing miR156f, the precursor of miR156, two transgenic lines miR156fOE...

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