Restoring pollen fertility in transgenic male-sterile eggplant by Cre/loxp-mediated site-specific recombination system

Abstract

This study was designed to control plant fertility by cell lethal gene Barnase expressing at specific developmental stage and in specific tissue of male organ under the control of Cre/loxP system, for heterosis breeding, producing hybrid seed of eggplant. The Barnase-coding region was flanked by loxP recognition sites for Cre-recombinase. The eggplant inbred/pure line ('E-38') was transformed with Cre gene and the inbred/pure line ('E-8') was transformed with the Barnase gene situated between loxp. The experiments were done separately, by means of Agrobacterium co-culture. Four T(0) -plants with the Barnase gene were obtained, all proved to be male-sterile and incapable of producing viable pollen. Flowers stamens were shorter, but the vegetative phenotype was similar to wild-type. Five T (0) -plants with the Cre gene developed well, blossomed out and set fruit normally. The crossing of male-sterile Barnase-plants with Cre expression transgenic eggplants resulted in site-specific excision with the male-sterile plants producing normal fruits. With the Barnase was excised, pollen fertility was fully restored in the hybrids. The phenotype of these restored plants was the same as that of the wild-type. Thus, the Barnase and Cre genes were capable of stable inheritance and expression in progenies of transgenic plants.

Keywords: Barnase gene; Cre gene; Cre/loxP system; eggplant; male sterility.

Figure 1

Sketch map of plant expression vector of Barnase and Cre gene. a: pCABARTABn. b: pBINPLUSCre.

Figure 2

Southern-blot analysis of transgenic plants. (a) Southern blot of Barnase transgenic plants; CK^_ showing a non-transgenic plant (E-8); B₃, B₇, B₁₅, B₃₆ showing Barnase transgenic plants; (b) Southern blot of Cre transgenic plants; CK^- showing a non-transgenic plant (E-38); R₁, R₂₇, R₄₀, R₅₂, R₆₃ showing Cre transgenic plants.

Figure 3

Northern blotting analysis of transgenic plants. (a) Cre gene expression in different parts of transgenic and non-transgenic plants; lanes 1-4 showing the flower, stem, leaf and root of a transgenic plant (R₆₃); lanes 5-8 showing the flower, stem, leaf and root of a non-transgenic plant(E-38). (b) Barnase gene expression in different parts of transgenic and non-transgenic plants; lanes 1-4 showing the root, stem, leaf and flower of a non-transgenic plant (E-8); lanes 5-8 showing the root, stem, leaf and flower of a transgenic plant (B₃); (c) Barnase gene expression in the flower of four different transgenic plants (B₃, B₇, B₁₅ and B₃₆); CK^-: a non-transgenic plant (E-8); (d) Cre gene expression in the leaf of five different transgenic plants (R₁, R₂₇, R₄₀, R₅₂ and R₆₃); CK^-: a non-transgenic plant (E-38).

Figure 4

Testing the viability of pollen from a Barnase transgenic plant(B₃) and a non-transgenic plant. (a) TTC testing of pollen from a nontransgenic plant (E-8). (b) TTC testing of pollen from a Barnase gene transgenic plant (B₃). (c) showing the germination of pollen from a nontransgenic plant (E-8). (d) showing the germination of pollen from a Barnase gene transgenic plant (B₃).

Figure 5

Stigmata, anthers and flowers of a Barnase transgenic plant and a non-transgenic plant. Panel1 showing the flower of a transgenic MS plant (B₇), 2 and 4 the flower of a transgenic MS plant (B₃), and 3 and 5 showing the flower of a non-transgenic plant (E-8).

Figure 6

PCR detection of Barnase-related MS and male-fertile plants in the progeny of a Barnase transgenic plant (B₃) crossed with a non-transgenic (E-8). M: Marker; lane 1 showing positive CK (pCABARTABn); lanes 2-14 showing male-sterile plants among progenies of B₃ x E-8; lanes 15-22 showing male fertile plants among progenies of B₃ x E-8.

Figure 7

Fruit in F₁ of a cross between a male-sterility plant (B₃) and a Cre-expressing plant (R₆₃).

Figure 8

PCR detection of progenies of Barnase , Bar and Cre in progenies of transgenic plant crossings. M: Markers; Ck⁺: showing positive CK (pCABARTABn + pBINPLUSCre); CK^_ showing a non-transgenic plant (E-8 x E-38); Lanes 1-9 showing a F₁ plant from crossing a transgenic male-sterile plant (B₃) and a transgenic plant with Cre gene (R₆₃).

Figure 9

Southern blotting for Barnase gene detection in F₁ of a crossing between a male-sterile plant (B₃) and a Cre-expressing plant (R₆₃). Lane Ck+ showing positive CK (Barnase gene PCR product); lane CK^_ showing a non-transgenic plant (E-8 x E-38); lanes 1-16 showing F₁ progeny from the crossing of a transgenic male-sterile plant (B₃) and a Cre transgenic plant (R₆₃).

Figure 10

Sequence structure and site characteristics before and after Cre-mediated recombination.

Figure 11

PCR analysis of the change in fragment size after recombination. M: λ DNA/EcoRV +Hind marker; lanes lane 1 showing negative control (F₁ of E-8 x E-38, non-transgenic plant); lane2 showing pCABARTABn; lane 3 showing a transgenic male-sterile plant (B₃); lanes 4-6 showing sterile gene-deleted F₁ plants (B₃ x R₆₃).